Production and use of purpurins, chlorins and purpurin- and chlorin-containing compositions

ABSTRACT

Families of chlorins, families of purpurins and metal complexes thereof are disclosed. The purpurins and their metal complexes have the structures of FIGS. 1, 7, 14-18, 29-38, 44-48 and 54-58 of the attached drawings. The chlorins and their metal complexes have the formulas of FIGS. 2, 8, 19, 20, 22, 23, 24, 25, 27, 28, 39, 40, 42, 43 and 49-53 of the attached drawings. Solutions of the purpurins, of the foregoing and other chlorins and of the metal complexes which are physiologically acceptable for intravenous administration are also disclosed, as are emulsions or suspensions of the solutions. The solvent for the solutions can be a product of the reaction of ethylene oxide with castor oil. A method for detecting and treating tumors in human and animal patients is also disclosed. The method comprises administering one of the purpurins, chlorins or metal complexes to the patient. For detection, the patient is then illuminated with ultra violet light; for treatment, the patient is illuminated with visible light of a wavelength at which the purpurin, chlorin or complex administered shows an absorption peak. 
     Families of purpurins, chlorins and metal complexes which can be detected by nuclear magnetic resonance or by an instrument that detects ionizing radiation are also disclosed. These compounds have the formula of one of FIGS. 1, 2, 7, 8, or 14-58 and a structure which is enriched in an atom that can be detected by nuclear magnetic resonance, e.g., C-13 or N-15, or by an instrument that detects ionizing radiation, e.g., C-14.

REFERENCE TO RELATED APPLICATIONS

This is continuation in part of application Ser. No. 07/388,643, filedAug. 2, 1989 (now U.S. Pat. No. 5,051,415 granted Sep. 4, 1991), whichwas in turn a continuation in part of application Ser. No. 874,097,filed Jun. 13, 1986, now abandoned and was also a continuation in partof application Ser. No. 842,125, filed Mar. 18, 1986, now abandoned, asa continuation in part of application Ser. No. 815,714, filed Jan. 2,1986, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to the production and use of a family ofpurpurins, a family of chlorins and metal complexes of the purpurins andchlorins, and to purpurin- and chlorin-containing compositions. Thepurpurins have a reduced pyrrole ring and an unsaturated isocyclic ringfused to a pyrrole ring; the unsaturated isocyclic ring of the purpurinscorresponds with a saturated ring in the chlorins. The chlorins areuseful as green dyes. The chlorins and the purpurins are useful in thedetection and treatment of tumors; after they have been administeredsystemically, e.g., intravenously, they localize preferentially in atumor. After they have been administered, and have localized in a tumor,their presence can be detected by illumination with ultra violet light,which causes them to fluoresce. The chlorins and purpurins of theinvention can also be used to treat tumors; after they have beenadministered and have localized, irradiation with light of a wave lengthat which they show an absorbance peak causes a reaction which damages ordestroys the tumor where they have localized. The purpurin- andchlorin-containing compositions are solutions thereof in an organicliquid that is physiologically acceptable for intravenous or topicaladministration and emulsions or suspensions of such solutions and wateror saline or other solutions.

2. The Prior Art

Four purpurins having an unsaturated isocyclic ring fused to a reducedpyrrole ring are known to be reported in the prior art, a communicationto the editor by Woodward et al., JACS, Vol. 82, pp. 3800 et seq., 1960,where they are disclosed as intermediates in the synthesis ofchlorophyll, and journal articles by Witte et al., Angew, Chem.Internat. Edit./Vol. 14, No. 5, pp. 361 et seq., 1975, and Arnold etal., Journal of the Chemical Society, Perkin Transactions I, pp. 1660 etseq., 1979. No utility for purpurins is suggested by either Witte et al.or Arnold et al. In addition European patent application EP142,732 (C.A.103: 123271S) discloses certain chlorins of a different family and thatthey accumulate preferentially in cancer cells removed from hamstersthat had been infected with pancreatic cancer.

Purpurins and chlorins are similar in structure to porphyrins. Oneporphyrin, called protoporphyrin IX, can be separated from blood.Hematoporphyrin can be produced from protoporphyrin IX; a chemicalmixture derived from hematoporphyrin, called hematoporphyrin derivative,and often abbreviated "HpD", can be administered intravenously and usedin the manner described above for the detection and treatment of tumors.The exact composition of HpD, however, is not known; in fact, it is amixture of many different porphyrins and related compounds (see, forexample, Porphyrin Photosensitization, edited by David Kassel and ThomasJ. Dougherty, Plenum Press, New York and London, 1983, pp. 3-13). As aconsequence, the chlorins and purpurins of the instant invention arepreferred over HpD for this use because they are single, knowncompounds. In addition, the chlorins and purpurins have absorbance peaksat longer wavelengths and show greater absorbances, by comparison withHpD; the longer wavelength peaks are advantageous because light of thelonger wavelengths is capable of greater penetration of tissue, whilethe greater absorbances are desirable because less light energy isrequired to cause a given degree of reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural formula for metal complexes of a family ofpurpurins in accordance with the instant invention; in these complexesan unsaturated isocyclic ring is fused to a reduced pyrrole ring.

FIG. 2 is a structural formula for metal complexes of a family ofchlorins in accordance with the instant invention; in these complexes;an isocyclic ring which corresponds with the unsaturated isocyclic ringof the purpurins of FIG. 1 is saturated.

FIG. 3 is a structural formula for a family of porphyrins which can beused to produce purpurins having the formula of FIG. 7.

FIG. 4 is a structural formula for pyrroles from which porphyrins havingthe structure of FIG. 3 can be produced.

FIG. 5 is a structural formula for dipyrromethanes which areintermediates for the production of porphyrins from pyrroles.

FIG. 6 is a structural formula for metal complexes of porphyrins havingthe formula of FIG. 3.

FIG. 7 is a structural formula for the family of purpurins having thestructure of the complexes of FIG. 1.

FIG. 8 is a structural formula of the family of chlorins having thestructure of the complexes of FIG. 2.

FIGS. 9-13 are structural formulas for five different carboxy purpurins,each of which can be used in the synthesis of different position isomersof purpurins having the structure of FIG. 7.

FIGS. 14-18, FIGS. 19-23, and FIGS. 24-28 are structural formulas forintermediates in the synthesis of purpurin position isomers having theformulas of FIGS. 29-33 from the carboxy purpurins having the formulasof FIGS. 9-13.

FIGS. 34-38 are structural formulas for purpurins according to theinvention having two isocyclic rings fused to pyrrole rings.

FIGS. 39-43 are structural formulas for chlorins, some of which areaccording to the invention, which can be produced from purpurins havingthe formulas of FIGS. 29-33.

FIGS. 44-48 are structural formulas for metal complexes of purpurinshaving the formulas of FIGS. 29-33.

FIGS. 49-53 are structural formulas for metal complexes of chlorinshaving the formulas of FIGS. 39-43.

FIGS. 54-58 are structural formulas for metal complexes of purpurinshaving the formulas of FIGS. 34-38.

BRIEF DESCRIPTION OF THE INVENTION

The instant invention, in one aspect, is a solution in an organic liquidof a purpurin, of a purpurin metal complex, of a chlorin or of a chlorinmetal complex. The purpurin has the structure of any of FIGS. 7, 14-18or 29-38 of the attached drawings; the purpurin metal complex has thestructure of any of FIGS. 1, 44-48 or 54-58, of the attached drawings,the chlorin has the structure of any of FIGS. 8, 19-28 or 39-43 of theattached drawings, and the chlorin metal complex has the structure ofany of FIGS. 2 or 49-53, of the attached drawings. In the drawings, Mrepresents a metal, for example, Ag, Al, Ce, Co, Cr, Dy, Er, Eu, Fe, Ga,Gd, Hf, Ho, In, La, Lu, Mn, Mo, Nd, Pb, Pd, Pr, Pt, Rh, Sb, Sc, Sm, Sn,Tb, Tc-99m, The, Ti, Tl, Tm, U, V, Y, Yb, Zn or Zr,

each of R10 through R13 and R16 is hydrogen, and each of R1 through R9,R14 and R15 is:

H or CHO,

an alkyl group other than t-butyl having from 1 to 4 carbon atoms,

an alkylene group having from 2 to 4 carbon atoms,

a group having the formula R₂ N(R₃)₂ where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; R₃ is hydrogen or an alkyl group having from 1 to2 carbon atoms and the two R₃ groups can be the same or different,

a group having the formula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; A is a physiologically acceptable anion; and R₄ isan alkyl group having from 1 to 2 carbon atoms and the three R₄ groupscan be the same or different;

a group having the formula R₂ OH were R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond,

an amino acid moiety which is a part of an amide produced by reactionbetween an amine function of a naturally occurring amino acid and acarboxyl function of the purpurin or chlorin,

CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R', where R' is hydrogen or an alkylgroup other than t-butyl having from 1 to 4 carbon atoms,

a monoclonal antibody moiety which is attached to the purpurin orchlorin moiety through a carbonyl which is a part of an amide producedby reaction between an amine function of a monoclonal antibody and a CO₂R' CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' group of the purpurin or chlorin, andwherein the moiety is of a monoclonal antibody which selectively bindsto malignant tumors, or

in the purpurins and purpurin metal complexes of FIGS. 14-18, 29-38,44-48 and 54-58 and in the chlorins and chlorin metal complexes of FIGS.24-28, 39-43 and 49-53 R1 can be a bivalent aliphatic hydrocarbonradical having from 2 to 4 carbon atoms wherein both of the valences ofthe radical are attached to the same carbon atom thereof and to a carbonatom of the purpurin, chlorin, or metal complex, and in the purpurinsand purpurin metal complexes of FIGS. 29-33 and of FIGS. 44-48, both R1and R2 can be bivalent aliphatic hydrocarbon radicals having from 2 to 4carbon atoms wherein both of the valences of the radical are attached tothe same carbon atom thereof and to a carbon atom of the purpurin ormetal complex, with the proviso that not more than one of R1 through R9,R14 and R15 is CHO, a group having the formula R₂ N(R₃)₂, a group havingthe formula R₂ N(R₄)₃ A, an amino acid moiety or a monoclonal antibodymoiety, and wherein the solution is one which is physiologicallyacceptable and of a suitable concentration or dilutable to a suitableconcentration for intravenous administration.

In another aspect, the invention is a chlorin or chlorin metal complexhaving the structure of any of FIGS. 2, 8, 19, 20, 22, 23, 24, 25, 27,28, 39, 40, 42, 43 and 49-53 of the attached drawings where R1 throughR16 and M have the meanings set forth above.

In yet another aspect, the invention is a method for detecting andtreating tumors which comprises administering an effective amount of apurpurin, chlorin or metal complex to a human or animal patient, andirradiating the relevant region of the patient with ultra violet or withvisible light of a wavelength at which the purpurin or chlorin has anabsorbance peak. The purpurin or purpurin complex is one having thestructure of any of FIGS. 1, 7, 14-18, 29-38, 44-48, or 54-58, while thechlorin or chlorin complex is one having the structure of any of FIGS.19, 20, 22, 23, 24, 25, 27, 29, 39, 40, 42, 43, and 49-53 of theattached drawings, where R1 through R16 and M have the meanings setforth above.

In still another aspect, the invention is a purpurin, a chlorin or ametal complex which has a structure that is enriched in an atom that canbe detected by nuclear magnetic resonance. The atom can be, for example,N-15 or C-13. The purpurin, chlorin or complex can have the formula ofany of FIGS. 1, 2, 7, 8 or 14-58 where M and R1 through R16 have themeanings set forth above.

In yet another aspect the invention is a method for the treatment ofnon-malignant lesions, e.g., of the vagina or bladder, or such cutaneouslesions as are involved in psoriasis; the method involves the topicalapplication of a chlorin, a purpurin or a complex, e.g., as a solutionin DMSO or ethanol, and illumination of the area in question. Thepurpurin, chlorin or complex can have the formula of any of FIGS. 1, 2,7, 8 or 14-58 where M and R1 through R16 have the meanings set forthabove.

In still another aspect, the invention is a method for treating human oranimal patients which involves administering a chlorin, purpurin ormetal complex and then treating the affected region with X rays. Thereare indications that the chlorins and the like are X ray sensitizerswhich increase the therapeutic ratio of X rays.

In another aspect, the invention is a chlorin, a purpurin or a complexmoiety coupled to a monoclonal antibody moiety. The moieties are formedwhen the antibody is reacted with the chlorin or the like, and, as aconsequence, the two are coupled through a carbonyl which is a part ofan amide produced by reaction between the monoclonal antibody and thepurpurin, chlorin or complex. The monoclonal antibody binds to a tumor,in this way increasing the selectivity of the chlorin or the likecoupled thereto. The purpurin, chlorin or complex can have the formulaof any of FIGS. 1, 2, 7, 8, or 14-58 where M is a one of the foregoingmetals, each of R10 through R13 and R16 is hydrogen, one of R1 throughR9, R14 and R15 is a monoclonal antibody which is an amino acid moietyattached to the purpurin or chlorin moiety through a carbonyl which is apart of an amide produced by reaction between the monoclonal antibodyand the purpurin or chlorin, and each of the others of R1 through R10,R14 and R15 has the meaning indicated above.

In still another aspect the invention is a purpurin, a chlorin or ametal complex which has a structure that is enriched in a radioactiveatom that can be detected by an instrument for measuring ionizingradiation. The atom can be, for example, C-14. The purpurin, chlorin orcomplex can have the formula of any of FIGS. 1, 2, 7, 8, or 14-58 whereM and R1 through R16 have the meanings set forth above.

In a final aspect the invention is a purpurin or a purpurin metalcomplex having the structure of any of FIGS. 1, 7, 14-18 or 29-38, 44-48or 54-58 of the attached drawings, where M and R1 through R16 have themeanings set forth above, except that R' is hydrogen or an alkyl groupother than t-butyl having from 2 to 4 carbon atoms.

OBJECTS OF THE INVENTION

It is, therefore, an object of the invention to provide a newcomposition which is a solution in an organic liquid of a purpurin or apurpurin complex having the structure of any of FIGS. 1, 7, 14-18,29-38, 44-48 or 54-58 or of a chlorin or a chlorin complex having thestructure of any of FIGS. 2, 8, 19-28, 39-43 or 49-53 of the attacheddrawings.

It is a further object to provide an aqueous emulsion of a solution inan organic liquid of a purpurin or purpurin complex having the structureof any of FIGS. 1, 7, 14-18, 29-38, 44-48 or 54-58 or of a chlorin orchlorin complex having the structure of any of FIGS. 2, 8, 19-28, 39-43or 49-53 of the attached drawings.

It is another object to provide a family of purpurins or purpurincomplexes having the structure of any of FIGS. 1, 7, 14-18, 29-38, 44-48or 54-58 of the attached drawings.

It is still another object to provide a family of chlorins or chlorincomplexes having the structure of any of FIGS. 2, 8, 19, 20, 22, 23-25,27, 28, 39, 40, 42, 43, or 49-53 of the attached drawings.

It is a further object to provide a purpurin, a chlorin or a metalcomplex which has a structure that is enriched in an atom that can bedetected by nuclear magnetic resonance.

It is still another object of the invention to provide a purpurin,chlorin or metal complex coupled to a monoclonal antibody.

It is a still further object to provide a method for the treatment ofnon-malignant lesions or of such cutaneous lesions as are involved inpsoriasis by the topical application of a solution in DMSO or the likeof one of the foregoing purpurins, chlorins or metal complexes, followedby illumination of the affected region.

It is yet another object of the invention to provide a method fordetecting and treating tumors which comprises administering one of theforegoing purpurins or chlorins to a human or animal patient, followedby illumination of the region affected with ultraviolet, with visiblelight or with X rays, scanning of the region affected by nuclearmagnetic resonance, or scanning of the region affected with aninstrument that measures ionizing radiation.

It is a still further object of the invention to provide a purpurin, achlorin or a metal complex which has a structure that is enriched in anatom that can be detected by an instrument that measures ionizingradiation.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Examples 1 through 3 and A through G hereof set forth the best modepresently contemplated by the inventors, insofar as this invention isdirected to purpurins, chlorins, metal complexes and their production.The in vivo test procedures describe the best mode insofar as thisinvention is directed to solutions of the purpurins and chlorins in anorganic liquid and to the production of such solutions, and the in vitroand in vivo test procedures describe the best mode insofar as theinvention is directed to the use of purpurins and chlorins for thedetection and treatment of tumors.

In the examples, and elsewhere herein, the term "percent v/v" meanspercent by volume; the term "percent w/w" means percent by weight; theterm "alkyl group" is used in its ordinary sense to mean a monovalent,saturated, aliphatic hydrocarbon radical; the term "alkylene group" isused in its ordinary sense to mean a monovalent, aliphatic hydrocarbonradical which has one carbon to carbon double bond and in which anyother carbon to carbon bond is a single bond; all temperatures are in°C.; and the following abbreviations have the meanings indicated:mg=milligram or milligrams; g=gram or grams; kg=kilogram or kilograms;ml=milliliter or milliliters; cm=centimeter or centimeters; ε=molarabsorptivity; and mw=milliwatts.

EXAMPLE 1

The production of a novel purpurin according to the invention (hereafter"Purpurin I") is described in this example. The synthesis involves theproduction of several pyrroles which are identified and assigned trivialnames in the following table:

    ______________________________________                                        Compound    Structure (referring to attached drawings)                        ______________________________________                                        Pyrrole I   FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is CO.sub.2 CH.sub.2 CH.sub.3                                               D is CH.sub.3                                            Pyrrole II  FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is CO.sub.2 CH.sub.2 CH.sub.3                                               D is CH.sub.2 OCOCH.sub.3                                Pyrrole III FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is COCH.sub.3                                                               D is CH.sub.3                                            Pyrrole IV  FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is CH.sub.2 CH.sub.3                                                        D is CH.sub.3                                            Pyrrole V   FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is CH.sub.2 CH.sub.3                                                        D is H                                                   Pyrrole VI  FIG. 4,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                                  B is CH.sub.2 CH.sub.3                                                        C is CH.sub.2 CH.sub.3                                                        D is CH.sub.2 OCOCH.sub.3                                ______________________________________                                    

The synthesis also involves the production of several dipyrromethaneswhich are identified and assigned trivial names in the following table:

    ______________________________________                                        Compound     Structure (referring to attached drawings)                       ______________________________________                                        Dipyrro-     FIG. 5,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5            methane I             B is CH.sub.2 CH.sub.3                                                        C is CO.sub.2 CH.sub.2 CH.sub.3                                               E is CH.sub.2 CH.sub.3                                                        F is CH.sub.2 CH.sub.3                                                        G is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                  Dipyrro-     FIG. 5,  where A is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5            methane II            B is CH.sub.2 CH.sub.3                                                        C is CH.sub.2 CH.sub.3                                                        E is CH.sub.2 CH.sub.3                                                        F is CH.sub.2 CH.sub.3                                                        G is CO.sub.2 CH.sub.2 C.sub.6 H.sub.5                  Dipyrro-     FIG. 5,  where A is CHO                                          methane III           B is CH.sub.2 CH.sub.3                                                        C is CH.sub.2 CH.sub.3                                                        E is CH.sub.2 CH.sub.3                                                        F is CH.sub.2 CH.sub.3                                                        G is CHO                                                ______________________________________                                    

The synthesis also involves the production of two porphyrins and sixporphyrin complexes, all of which are identified and given trivial namesin the following table:

    ______________________________________                                        Compound  Structure (referring to attached drawings)                          ______________________________________                                        Porphyrin I                                                                             FIG. 3,  where R and R10-R12 are hydrogen                                              R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                           Porphyrin FIG. 6,  where R and R10-R12 are hydrogen                           Complex II         R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              M is Ni                                                    Porphyrin FIG. 6,  where R is CHO                                             Complex III        R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R10-R12 are H                                                                 M is Ni                                                    Porphyrin FIG. 6,  where R, R11 and R12 are H                                 Complex IV         R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R10 is CHO                                                                    M is Ni                                                    Porphyrin FIG. 6,  where R, R10 and R12 are H                                 Complex V          R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R11 is CHO                                                                    M is Ni                                                    Porphyrin FIG. 6,  where R, R10 and R11 are hydrogen                          Complex VI         R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R12 is CHO                                                                    M is Ni                                                    Porphyrin FIG. 6,  where R, R10 and R12 are hydrogen                          Complex VII        R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R11 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                                    M is Ni                                                    Porphyrin VIII                                                                          FIG. 3,  where R, R10 and R12 are hydrogen                                             R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R11 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                 ______________________________________                                    

Purpurin I and other purpurins that were produced in the course of thesynthesis thereof are identified and the other purpurins are assignedtrivial names in the following table:

    ______________________________________                                        Compound Structure (referring to attached drawings)                           ______________________________________                                        Purpurin I                                                                             FIG. 34: where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3                                  R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R13 is H                                                                      R15 is CO.sub.2 CH.sub.2 CH.sub.3                           Purpurin II                                                                            FIG. 9:  where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3                                  R9 is CO.sub.2 CH.sub.2 CH.sub.3                            Purpurin III                                                                           FIG. 13: where R1-R6 and R8 are CH.sub.2 CH.sub.3                                      R9 is CO.sub.2 CH.sub.2 CH.sub.3                            Purpurin IV                                                                            FIG. 14: where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3                                  R9 is CO.sub.2 CH.sub.2 CH.sub.3                            Purpurin V                                                                             FIG. 7:  where R1-R5, R7 and R8 are CH.sub.2 CH.sub.3                                  R6 is (O═C)CH.sub.2 CO.sub.2 CH.sub.3                                     R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R10-R13 are hydrogen                                        ______________________________________                                    

PRODUCTION OF PYRROLE I

Pyrrole I is produced from a saturated aqueous solution containing onegram equivalent of sodium nitrite, a 5 percent w/w solution in glacialacetic acid containing one gram equivalent of benzyl propionylacetate, asuspension in glacial acetic acid of one gram equivalent of ethylacetoacetate and four gram equivalents of zinc dust. The sodium nitritesolution is added dropwise, with stirring, to the benzylpropionylacetate solution at a rate sufficiently slow that thetemperature of the solution that is formed does not exceed 20. After theresulting solution stands at room temperature of about 22 for thirtyminutes, the zinc dust is added in increments to the ethyl acetoacetatesuspension at such a rate that the exothermic reaction which occursheats the slurry to about 65; the foregoing solution is then addeddropwise, with stirring, to the ethyl acetoacetate/zinc slurry. Thereaction mixture is maintained at 65 during the addition and for an hourafter completion of the addition of the sodium nitrite-benzylpropionylacetate solution; stirring is continued during the additionalhour. The hot solution is then separated from the zinc by decantation.The Pyrrole I is precipitated by pouring the hot solution into icewater, and is recovered by filtration and air dried.

PRODUCTION OF PYRROLE II

Pyrrole II is produced from 6 g lead tetra-acetate and a solution of 4 gPyrrole I in 2 ml acetic anhydride and 100 ml glacial acetic acid. Thelead tetra-acetate is added to the Pyrrole I solution and dissolved bywarming the resulting reaction mixture on a steam bath for about 10minutes. The solution so formed is stirred at room temperature of about22 for 16 hours. Dropwise additions of ice are then made to precipitatethe Pyrrole II, which is recovered from the reaction mixture byfiltration and washed with distilled water.

PRODUCTION OF PYRROLE III

Pyrrole III is produced from a saturated aqueous solution containing onegram equivalent of sodium nitrite, a 5 percent w/w solution in glacialacetic acid containing one gram equivalent of benzyl propionyl-acetate,a suspension in glacial acetic acid of one gram equivalent of2,4-pentanedione and four gram equivalents of zinc dust. The sodiumnitrite solution is added dropwise, with stirring, to the benzylpropionyl-acetate solution at a rate sufficiently slow that thetemperature of the solution that is formed does not exceed 20. After theresulting solution stands at room temperature of about 22 for thirtyminutes, the zinc dust is added in increments to the 2,4-pentanedionesuspension at such a rate that the exothermic reaction which occursheats the slurry to about 65; the foregoing solution is then addeddropwise, with stirring, to the 2,4-pentanedione/zinc slurry. Thereaction mixture is maintained at 65 during the addition and for an hourafter completion of the addition of the sodium nitrite/benzylpropionyl-acetate solution; stirring is continued during the additionalhour. The hot solution is then separated from the zinc by decantation.The Pyrrole III is precipitated by pouring the hot solution into icewater, and is recovered by filtration and air dried.

PRODUCTION OF PYRROLE IV

Pyrrole IV is produced from 1.1 gram equivalents of boron trifluorideetherate and a 5 percent w/w solution in glacial acetic acid containingone gram equivalent of Pyrrole III; the Pyrrole III solution alsocontains 2 gram equivalents of sodium borohydride. The Pyrrole IIIsolution is cooled with ice while the boron trifluoride etherate isadded thereto dropwise, with stirring. After the addition is complete,the reaction mixture is allowed to warm to room temperature of about 22and then to stand two hours at room temperature; stirring is continuedthroughout. Excess sodium borohydride is then destroyed by cautiousadditions of glacial acetic acid. The reaction mixture, a solution, isthen poured into ice water; Pyrrole IV which precipitates is recoveredby filtration and air dried.

PRODUCTION OF PYRROLE V

Pyrrole V is produced from a 5 percent w/w solution in dichloromethanecontaining 1 gram equivalent of Pyrrole IV and a 5 percent w/w solutionin dichloromethane containing 4 gm equivalents of sulfuryl chloride. ThePyrrole IV solution is diluted with about 10 percent v/v diethyl etherand the sulfuryl chloride solution is added to the diluted Pyrrole IVsolution. The reaction is conducted at room temperature with stirring,which is commenced before the sulfuryl chloride solution addition isstarted, and continued for about one hour after that addition iscompleted. Solvent is then removed from the reaction mixture, leaving apale yellow oil. The oil is dissolved in a solution of water in acetonecontaining 20 percent v/v water and the solution is heated under reflux.The solution becomes acidic rapidly. After 20 minutes under reflux,enough sodium acetate to neutralize the acid and a small excess is addedto the solution; heating is continued until the acetone is vaporized andan oil separates from the aqueous phase which remains. Upon cooling ofthe reaction mixture to room temperature, the oil forms a crystallizedsolid ("Pyrrole VII": Pyrrole V, except that D is CO₂ H) which issuspended in a 5 percent w/w solution in glacial acetic acid and 4.8percent w/w acetic anhydride containing 3 equivalents of anhydroussodium acetate per equivalent of the Pyrrole VII. The resultingsuspension is heated gently to 80 and stirred with a 5 percent w/wsolution in glacial acetic acid containing 1 equivalent of iodinemonochloride (based upon the Pyrrole VII) is added dropwise thereto.When the iodine monochloride addition is complete, the solution whichhas formed is cooled and mixed with an equal volume of water;hypophosphorous acid is added to remove excess iodine; and the solidwhich forms is recovered by filtration, washed with water, dried andsuspended with 0.2 g platinum oxide in tetrahydrofuran. The resultingsuspension is hydrogenated until the uptake of hydrogen ceases. Thesolution of Pyrrole V in tetrahydrofuran which results is separated fromthe platinum oxide by filtration; the tetrahydrofuran is replaced underreduced pressure with methanol; and the Pyrrole V is recovered byfiltration.

PRODUCTION OF DIPYRROMETHANE I

Dipyrromethane I is produced from a 5 percent w/w solution indichloromethane containing one g equivalent Pyrrole II, one g equivalentPyrrole V and about 2 g Montmorillonite clay. The clay is added to thePyrrole II solution, and the slurry which results is stirred for about10 minutes. The clay is then separated by filtration and washed withdichloromethane; the wash is combined with the filtration; evaporationof the dichloromethane leaves the Dipyrromethane I.

PRODUCTION OF PYRROLE VI

Pyrrole VI is produced from 6 g lead tetra-acetate and a solution of 1 gequivalent Pyrrole IV in 2 ml acetic anhydride and 100 ml glacial aceticacid. The lead tetra-acetate is added to the Pyrrole II solution, andthe resulting reaction mixture is warmed on a steam bath to dissolve thelead tetra-acetate. The solution which is formed is stirred for 16 hoursat room temperature of about 22, after which time the Pyrrole VI isprecipitated by dropwise addition of ice water, separated from theliquid by filtration, washed with water and air dried.

PRODUCTION OF DIPYRROMETHANE II

Dipyrromethane II is produced by dissolving Pyrrole VI in methanolcontaining about 0.05 percent w/w HCl to make a 5 percent w/w solution,and heating the solution under reflex for five hours. The reactionproduct is cooled to room temperature of about 22 and poured into icewater to precipitate the Dipyrromethane II, which is then recovered byfiltration and air dried.

PRODUCTION OF DIPYRROMETHANE II

Dipyrromethane III is produced from 2 g Dipyrromethane II, 50 mlabsolute ethanol containing 0.05 percent w/w triethyl amine, 0.1 gcharcoal coated with 5 percent w/w palladium, 3 ml trifluoroacetic acidand 1.0 g p-nitobenzoyl chloride dissolved in 1.0 g dry dimethylformamide. The Dipyrromethane II is dissolved in the absolute ethanoland the palladium on charcoal is added to the resulting solution. TheDipyrromethane II is then hydrogenated in a sloping manifoldhydrogenator in which a slight positive pressure of hydrogen ismaintained until there is no longer an uptake of hydrogen. The palladiumon charcoal is then separated from the reaction mixture by filtration,and the solvent is evaporated, leaving a solid white residue. The whiteresidue is powdered finely and added, under nitrogen, to thetrifluoroacetic acid at a temperature of 45; the reaction mixture ismaintained at 45, with stirring, until the evolution of CO₂ subsides andfor an additional 3 minutes, and is then poured into 30 ml 56 percentw/w aqueous ammonium hydroxide to which 5 g crushed ice has been added.The aqueous mixture which results is extracted with dichloromethane,which is then evaporated; the red oil which remains after evaporation ofthe dichloromethane is immediately dissolved in 3 ml drydimethylformamide, and the solution which results is cooled to andmaintained at 0, with stirring, while the solution of p-nitrobenzoylchloride in dimethyl formamide is added dropwise, and for 30 minutesafter completion of the addition. An addition of 20 ml diethyl ether ismade 15 minutes after completion of the addition of the dimethylformamide solution, and, 15 minutes later, solids which haveprecipitated are separated from the liquid by filtration and added, withstirring, to 50 percent w/w aqueous ethanol containing about 3 g sodiumcarbonate, which has been heated to 70. After 15 minutes of stirring,Dipyrromethane III is separated from the ethanol/water solution byfiltration and air dried.

PRODUCTION OF PORPHYRIN I

Porphyrin I is produced from two solutions, one a 5 percent w/w solutionin dry tetrahydrofuran containing 0.05 percent w/w triethyl amine and 1g equivalent Dipyrromethane I, and the second a 5 percent w/w solutionin dichloromethane containing 5 percent v/v methanol, 0.05 percent w/wp-toluenesulfonic acid and 1 g equivalent of Dipyrromethane III, using 5percent w/w, based on the weight of the Dipyrromethane I, 5 percent w/wpalladium on charcoal as a hydrogenation catalyst. The palladium oncharcoal is added to the Dipyrromethane I solution and hydrogenation iscarried out in a sloping manifold hydrogenator in which a slightpositive pressure of hydrogen is maintained until the uptake of hydrogenstops. The palladium on charcoal is then separated from the reactionmixture by filtration and washed with dilute ammonium hydroxide. Thefiltrate is evaporated to dryness in vacuo, and the product whichremains is dissolved in the ammonia washings; 12 percent w/w acetic acidis added to the resulting solution to adjust the pH to 4 and thetemperature thereof is lowered to about 5 to cause precipitation of anintermediate diacid. The diacid is then dissolved in the second solutionand the resulting reaction mixture is allowed to stand in the dark atroom temperature of about 22° for 24 hours, after which time amethanolic solution containing about 1 g zinc acetate dihydrate is addedthereto. The solution is allowed to stand in the dark at roomtemperature for another 72 hours, after which time the solvent isremoved by evaporation, and the solid which remains is dissolved inaqueous dioxane containing 3 equivalents KOH per equivalent of PorphyrinI. The resulting solution is refluxed for four hours, cooled, anddiluted with distilled water; the Porphyrin I is extracted from thesolution with dichloromethane; the dichloromethane is evaporated; andthe residue is recrystallized from a 50 percent v/v solution of methanolin dichloromethane.

PRODUCTION OF PORPHYRIN COMPLEX II

A 5 percent w/w solution in a mixed dichloromethane-methanol solventcontaining 20 percent v/v methanol, 1 g equivalent of Porphyrin I and 2g equivalents of nickel acetate is refluxed for 16 hours. The solvent isthen evaporated until the Porphyrin Complex II precipitates; the productis recovered by filtration and air dried.

PRODUCTION OF PORPHYRIN COMPLEXES III-VI

A mixture of the porphyrin complexes identified above is prepared from 1g Porphyrin Complex II, 28 ml freshly distilled phosphorus oxychloride,20 ml dry dimethyl formamide and 750 ml dry 1,2-dichloroethane. Thedimethyl formamide is cooled on an ice bath, and the phosphorusoxychloride is added thereto dropwise. The solution which results isallowed to stand at room temperature of about 22° for 30 minutes, and isthen warmed to 50°. The Porphyrin Complex II is dissolved in the1,2-dichloroethane, and the resulting solution is added dropwise, withstirring, to the phosphorus oxychloride; the addition is made over aperiod of about 30 minutes. The reaction mixture is maintained at about50°, with stirring, for an additional 2 hours. The organic and theaqueous phases are then separated, and the aqueous phase is extractedwith dichloromethane. The organic phase and the dichloromethane extractare then combined, and evaporated to dryness. The solid which remains isrecrystallized from a solvent composed of equal parts by volume ofdichloromethane and methanol, yielding a mixture of Porphyrin ComplexIII, Porphyrin Complex IV, Porphyrin Complex V and Porphyrin Complex VI.The mixture of complexes is separated by silica gel chromatography,using dichloromethane containing 1 percent v/v methanol as the eluant.

PRODUCTION OF PORPHYRIN COMPLEX VII

A solution of 506 mg Porphyrin Complex V and 1.024 g.(carbethoxymethylene)triphenylphosphorane in 50 ml xylene is heatedunder reflux for 18 hours. The solution is cooled, the xylene is removedin vacuo; and the solid which remains is dissolved in the minimum amountof dichloromethane and chromatographed on silica gel. A minor fractionof Porphyrin Complex V and a major red fraction are recovered. Thesolvent is removed from the red fraction; the solid which remains isrecrystallized from a solvent composed of equal parts by volume ofdichloromethane and methanol, yielding Porphyrin Complex VII.

PRODUCTION OF PORPHYRIN VIII

A solution is prepared by dissolving 621 mg Porphyrin Complex VII in 10ml concentrated (96.7 percent w/w) sulfuric acid; after the solutionstands for 2 hours at room temperature of about 22°, an addition of 100ml dichloromethane is made thereto, followed by saturated aqueous sodiumbicarbonate to neutralize the sulfuric acid. The organic layer iscollected, washed and dried; the solvent is then vaporized. The crudeproduct which remains is recrystallized from a solvent composed of equalparts by volume of dichloromethane and methanol, yielding PorphyrinVIII.

PRODUCTION OF PURPURIN II AND PURPURIN III

A solution of 100 mg Porphyrin VIII in 20 ml glacial acetic acid isheated under reflux in a nitrogen atmosphere for 24 hours. The solutionis then cooled; the acetic acid is removed in vacuo; and the remainingproduct is dissolved in the minimum amount of dichloromethane andchromatographed on silica gel, yielding a major green fraction fromwhich the solvent is removed. The solid which remains is recrystallizedfrom 50 percent v/v dichloromethane and methanol, yielding a mixture ofPurpurin II and Purpurin III which are separated by silica gelchromatography using dichloromethane containing 1 percent v/v methanolas the elutant.

PRODUCTION OF PURPURIN V

Purpurin V is produced from 59 mg Purpurin II, 25 mgN,N'-carbonyldiimidazole, 100 mg zinc acetate, 25 mg sodium hydride and87 mg methyl t-butyl malonate. The Purpurin II is dissolved in 5 mldichloromethane and refluxed for one hour with theN,N'-carbonyldiimidazole; the zinc acetate dissolved in 5 ml methanol isthen added and the resulting reaction mixture is warmed gently for 5minutes. After an addition of 25 ml dichloromethane, the solution whichforms is washed three times with 50 ml portions of water, dried overMgSO₄ and evaporated under vacuum; the solid residue which remains afterevaporation of the solvents is maintained at an absolute pressure of 0.1mm Hg for 30 minutes, and is then dissolved in dichloromethane. Theresulting solution is then added to a malonate anion solution preparedby adding the sodium hydride and the methyl t-butyl malonate to 10 mltetrahydrofuran, and the reaction mixture is stirred for 40 minutes atroom temperature of about 22° and added to 50 ml chloroform and 20 ml 1normal hydrochloric acid. The organic phase is separated from theaqueous phase, washed twice with 50 ml portions of water, dried overMgSO₄ and evaporated; the residue is stirred at room temperature ofabout 22° with 5 ml trifluoroacetic acid for 40 minutes and theresulting product is mixed with 100 ml water and 50 ml chloroform. Theorganic layer is separated from the aqueous layer, washed twice with 50ml portions of water, dried over MgSO₄, and evaporated to dryness. Theresidue is dissolved in the minimum amount of dichloromethane containing5 percent v/v acetone and purified by elutriation on alumina. Purpurin Vis recovered by evaporating the solvents from the elutriate.

PRODUCTION OF PURPURIN IV

Purpurin IV is prepared from 68 mg Purpurin V and 110 mg thalliumtrifluoroacetate. A solution of the Purpurin V in 20 ml drydichloromethane and 20 ml dry tetrahydrofuran is treated with a solutionof the thallium trifluoroacetate in 10 ml dry tetrahydrofuran. After 2minutes, the solution which results is placed in sunlight for about 10minutes until a sample examined spectrophotometrically shows theexpected shift of the Soret absorption band. The solution is thentreated briefly with SO₂ gas, stirred for about 1 minute with about 1/2ml 37 percent w/w hydrochloric acid, diluted with 50 ml dichloromethane,and washed three times with 100 ml portions of water. The solvents arethen removed by evaporation; the residue is dissolved in dichloromethanecontaining 5 percent v/v methanol; the solution is chromatographed onalumina; and the Purpurin V is crystallized from the solvent andrecovered by filtration.

PRODUCTION OF PURPURIN I

A solution of 60 mg sodium borohydride in 10 ml methanol is addeddropwise to a solution of 200 mg Purpurin IV in 5 ml dichloromethane;the resulting solution is stirred at room temperature of about 22° for 2hours, and is poured into 100 ml water. The organic phase is separatedfrom the aqueous phase; the solvent is removed from the organic phase;and the solvent is evaporated. The residue is dissolved in 50 mlchloroform containing 25 percent v/v methanol; a 10 mg addition ofp-toluene sulfonic acid is made; and the reaction mixture is refluxedfor 6 hours. Water is then added to the reaction mixture; the organiclayer is collected; and the solvent is removed by evaporation. Theresidue is dissolved in 5 ml dichloromethane containing 2 percent v/vmethanol; the resulting solution is chromatographed on silica gel; andPurpurin I is recovered by evaporating the solvent from thechromatographed solution.

Purpurin II and Purpurin III are both produced from Porphyrin VIII inone step of the procedure described above as Example 1; Purpurin V,Purpurin IV and Purpurin I are then produced from Purpurin II. It willbe appreciated that Purpurin III can be substituted for Purpurin II toproduce the following isomers of Purpurin I, Purpurin IV and Purpurin V:

    ______________________________________                                        Compound   Structure (referring to attached drawings)                         ______________________________________                                        Purpurin VI                                                                              FIG. 38: where R1-R6 and R8 are CH.sub.2 CH.sub.3                                      R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R13 is H                                                                      R15 is CO.sub.2 CH.sub.2 CH.sub.3                         Purpurin VII                                                                             FIG. 18: where R1-R6 and R8 are CH.sub.2 CH.sub.3                                      R9 is CO.sub.2 CH.sub.2 CH.sub.3                          Purpurin VIII                                                                            FIG. 7:  where R1-R6 and R8 are CH.sub.2 CH.sub.3                                      R7 is (O═C)CH.sub.2 CO.sub.2 CH.sub.3                                     R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R10-R13 are hydrogen                                      ______________________________________                                    

Similarly, Porphyrin Complex III, Porphyrin Complex IV, PorphyrinComplex V and Porphyrin Complex VI are all produced in one step of theprocedure; Porphyrin Complex VII, Porphyrin VIII, Purpurin II andPurpurin III are then produced from Porphyrin Complex V. It will beappreciated that Porphyrin Complex IV can be substituted for PorphyrinComplex V to produce the following isomers of Porphyrin Complex VII,Porphyrin VIII, Purpurin I, Purpurin II, Purpurin IV and Purpurin V:

    ______________________________________                                        Compound Structure (referring to attached drawings)                           ______________________________________                                        Porphyrin                                                                              FIG. 6:  where R, R11 and R12 are hydrogen                           Complex IX        R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R10 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                                    M is Ni                                                     Porphyrin X                                                                            FIG. 3:  where R, R11 and R12 are hydrogen                                             R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R10 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                  Purpurin IX                                                                            FIG. 35: where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                                   R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R13 is H                                                                      R15 is CO.sub.2 CH.sub.2 CH.sub.3                           Purpurin X                                                                             FIG. 10: where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                                   R9 is CO.sub.2 CH.sub.2 CH.sub.3                            Purpurin XI                                                                            FIG. 15: where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                                   R9 is CO.sub.2 CH.sub.2 CH.sub.3                            Purpurin XII                                                                           FIG. 7:  where R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                                   R4 is (O═C)CH.sub.2 CO.sub.2 CH.sub.3                                     R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R10-R13 are hydrogen                                        ______________________________________                                    

Similarly, it will be appreciated that Porphyrin Complex VI can besubstituted for Porphyrin Complex V to produce the following isomers ofPorphyrin Complex VII, Porphyrin VIII and Purpurin II:

    ______________________________________                                        Compound  Structure (referring to attached drawings)                          ______________________________________                                        Porphyrin FIG. 6:  where R, R10 and R11 are hydrogen                          Complex XI         R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R12 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                                    M is Ni                                                    Porphyrin XII                                                                           FIG. 3:  where R, R10 and R11 are hydrogen                                             R1 and R3-R8 are CH.sub.2 CH.sub.3                                            R2 is CO.sub.2 H                                                              R12 is CH═CHCO.sub.2 CH.sub.2 CH.sub.3                 Purpurin XIII                                                                           FIG. 11: where R1-R7 are CH.sub.2 CH.sub.3                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                           Purpurin XIV                                                                            FIG. 12: where R1-R4 and R6-R8 are                                                     CH.sub.2 CH.sub.3                                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                           ______________________________________                                    

In a like manner, Purpurins XIII and XIV can be substituted for PurpurinII to produce other isomers of Purpurin I:

    ______________________________________                                        Compound     Structure (referring to attached drawings)                       ______________________________________                                        Purpurin XV  FIG. 36: where R1-R7 are CH.sub.2 CH.sub.3                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R13 is H                                                                      R15 is CO.sub.2 CH.sub.2 CH.sub.3                       Purpurin XVI FIG. 16: where R1-R7 are CH.sub.2 CH.sub.3                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                        Purpurin XVII                                                                              FIG. 7:  where R1-R7 are CH.sub.2 CH.sub.3                                             R8 is (O═C)CH.sub.2 CO.sub.2 CH.sub.3                                     R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R10-R13 are hydrogen                                    Purpurin XVIII                                                                             FIG. 37: where R1-R4 and R6-R8 are                                                     CH.sub.2 CH.sub.3                                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R13 is H                                                                      R15 is CO.sub.2 CH.sub.2 CH.sub.3                       Purpurin XIX FIG. 17: where R1-R4 and R6-R8 are                                                     CH.sub.2 CH.sub.3                                                             R9 is CO.sub.2 CH.sub.2 CH.sub.3                        Purpurin XX  FIG. 7:  where R1-R4 and R6-R8 are                                                     CH.sub.2 CH.sub.3                                                             R5 is (O═C)CH.sub.2 CO.sub.2 CH.sub.3                                     R9 is CO.sub.2 CH.sub.2 CH.sub.3                                              R10-R13 are hydrogen                                    ______________________________________                                    

It will be appreciated that other purpurins having the structures ofFIGS. 7, 9-18, 29-33 and 34-38 can be produced by the method of Example1 from porphyrins having an appropriate structure, if available, orsynthesized from dipyrromethanes having an appropriate structure, ifavailable; further, the requisite dipyrromethanes can be synthesized bythe method set forth from available pyrroles or from pyrrolessynthesized as described. Purpurins so produced have the structure ofone of the indicated figures of the drawings where each of R1 through R8is H,

a primary or secondary alkyl group having from 1 to 4 carbon atoms,

an alkylene group having from 2 to 4 carbon atoms,

a group having the formula R₂ N(R₃)₂ where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; R₃ is hydrogen or an alkyl group having from 1 to2 carbon atoms and the two R₃ groups can be the same or different,

a group having the formula R₂ N(R₄)₃ ⁺ where R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond; and R₄ is an alkyl group having from 1 to 2 carbonatoms and the three R₄ groups can be the same or different,

a group having the formula R₂ OH were R₂ is a bivalent aliphatichydrocarbon radical having from 1 to 4 carbon atoms, wherein any carbonto carbon bond is either a single or a double bond, and not more thanone is a double bond, or

CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' where R' is H, or a primary orsecondary alkyl group having from one to four carbon atoms.

In the purpurins so produced, R9 and R15 are usually CO₂ CH₃ or CO₂ CH₂CH₃, their identify being determined by that of the precursor porphyrin(see steps of producing Porphyrin Complex VII, Porphyrin VIII, andPurpurins II and III in Example 1; the identify of the CO₂ CH₂ CH₃groups in Purpurin II and in Purpurin III was determined by that of the-ethoxycarbonylvinyl moiety in Porphyrin VIII), and R10 through R14 arehydrogen.

Where any of R1 through R16 of any of the foregoing purpurins is CO₂ H,that moiety can be reacted with an amino acid moiety, which can be amonoclonal antibody, to form an amide. Example 2 is illustrative of suchreactions:

EXAMPLE 2

A purpurin coupled to a monoclonal antibody is produced form 20 mgPurpurin II dissolved in 1.25 ml water and 0.8 ml N,N-dimethylformamide, 20 mg 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide.HCldissolved in 0.6 ml water and 15 mg monoclonal antibody dissolved in 5ml distilled water. The Purpurin II solution is added to thecarbodiimide hydrochloride solution, and the combined solution is mixedwith the monoclonal antibody solution. After 30 minutes, the reaction isquenched by adding 0.05 ml monoethanol amine, and the conjugatedmaterial, i.e., the amide of the monoclonal antibody and Purpurin II, isdialyzed exhaustively at 4° against 0.001N phosphate buffered saline, pH7.4.

The procedure of Example 2 can be used to couple other proteins, otheramino acids, to Purpurin II and to other purpurins having a CO₂ H group.

It is known that some monoclonal antibodies, after they have beenadministered to a patient, for example intravenously, localize in tumorcells, specifically in malignant tumor cells, It is also known that somepurpurins and some chlorins, after administration, localize in a similarmanner and can then be detected with ultra violet light, which causesthem to fluoresce, or illuminated with visible light of a wavelength atwhich they show an absorbance peak, which causes them to undergo areaction which destroys the tumor in which they have localized; as issubsequently demonstrated in Examples 3-9 hereof, this is also true ofseveral purpurins having the formula of FIG. 7 of the attached drawingsand of several chlorins having the formula of FIG. 8. It will beappreciated, therefore, that a purpurin or chlorin with a monoclonalantibody which localizes in this way chemically attached thereto has anenhanced capability to localize and the same capability for detectionand destruction of a tumor where it has localized, by comparison withthe parent purpurin or chlorin.

EXAMPLE 3

The production of a purpurin (hereafter "Purpurin NT2") from nickelmeso-formyl octaethyl porphyrin is described in this Example. Theproduction of nickel meso-formyl octaethyl porphyrin is described in ajournal article by R. Grigg et al., J. Chem. Soc. Perkin Trans I, 1972,pp., 1789, 1798; it has the formula of FIG. 6 of the attached drawingswhere R1 through R8 are ethyl, R is CHO, R10 through R12 are hydrogen,and M is Ni. Two intermediates were produced in the Example 3 procedure,nickel Meso-(-ethoxycarbonylvinyl)octaethyl porphyrin, which has theformula of FIG. 6 of the attached drawings where R1 through R8 areethyl, R is CH═CHCO₂ CH₂ CH₃, R10 through R12 are hydrogen, and M is Ni,and meso(-ethoxycarbonylvinyl)octaethyl porphyrin, which has the formulaof FIG. 3 of the attached drawings where R1 through R8 are ethyl, R isCH═CHCO₂ CO₂ CH₃, and R10 through R12 are hydrogen. Purpurin NT2 has theformula of FIG. 7 of the attached drawings where R1 through R8 areethyl, R9 is CO₂ CH₂ CH₃, and R10 through R13 are hydrogen.

Production of Nickel Meso-(-ethoxycarbonylvinyl)octaethyl Porphyrin

A solution of 506 mg nickel meso-formyl octaethyl porphyrin and 1.024 g(carbethoxymethylene)triphenylphosphorane in 50 ml xylene was heatedunder reflux for 18 hours. The solution was cooled; the xylene wasremoved in vacuo; and the solid which remained was dissolved in theminimum amount of dichloromethane and chromatographed on silica. A minorfraction of nickel octaethyl porphyrin and a major red fraction wererecovered. The solvent was removed from the red fraction; the solidwhich remained was recrystallized from a solvent composed of equal partsby volume of dichloromethane and methanol, yielding 455 mg small brownneedles. The product was identified by nuclear magnetic resonance asnickel meso-(-ethoxycarbonylvinyl)octaethyl porphyrin; it showed visiblespectrum absorbance peaks at 405, 530 and 565 nanometers (94, 180, 18604, 27 790).

Production of Meso-(-ethoxycarbonylvinyl)octaethyeyl Porphyrin

A solution was prepared by dissolving 621 mg nickelmeso-(-ethoxycarbonylvinyl)octaethyl porphyrin in 10 ml concentrated(96.7 percent w/w) sulfuric acid; after the solution stood for 2 hoursat room temperature of about 22°, an addition of 100 ml dichloromethanewas made thereto, followed by saturated aqueous sodium bicarbonate toneutralize the sulfuric acid. The organic layer was collected, washedand dried; the solvent was then vaporized. The crude product whichremained was recrystallized from a solvent composed of equal parts byvolume of dichloromethane and methanol, yielding 552 mg smallreddish-brown crystals which were identified by nuclear magneticresonance as meso-(-ethoxycarbonylvinyl)-octaethyl porphyrin. Theproduction of this porphyrin is disclosed in a Journal article byFuhrhop et al., Ann. Chem., 1976, pp. 1539-1559.

Production of Purpurin NT2

A solution of 1090 mg meso-(-ethoxycarbonylvinyl)octaethyl porphyrin in20 ml glacial acetic acid was heated under reflux in a nitrogenatmosphere for 24 hours. The solution was then cooled; the acetic acidwas removed in vacuo; and the remaining product was dissolved in theminimum amount of dichloromethane and chromatographed on silica,yielding a major green fraction from which the solvent was removed. Thesolid which remained was recrystallized from 50 percent v/vdichloromethane and methanol yielding 68 mg purple microcrystals whichwere identified by nuclear magnetic resonance as Purpurin NT2, and foundto have visible spectrum absorbance peaks at 433, 453, 503, 530, 568,648 and 695 nanometers (89 509, 89 509, 14 571, 12 143, 18 908, 10 582,42 673).

EXAMPLE 4 Production of Zn Purpurin NT2

A solution was prepared by dissolving 20 mg Purpurin NT2 in a mixedsolvent composed of 15 ml dichloromethane and 5 ml methanol and 100 mgzinc acetate was added to the solution; the mixture which resulted wasrefluxed for about 4 minutes until the electronic spectrum of thereaction mixture indicated that chelation was complete. The reactionmixture was then concentrated to 7 ml and allowed to cool to roomtemperature at about 22°. Product which precipitated was recovered byfiltration, dissolved in a mixed solvent composed of 5 mldichloromethane and 2 ml methanol, and recrystallized, yielding 18 mg ZnPurpurin NT2 in the form of microcrystals. The Zn Purpurin NT2, a metalcomplex, has the formula of FIG. 1 of the attached drawings where R1through R8 are ethyl, R9 is CO₂ CH₂ CH₃, R10 through R13 are hydrogenand M is Zn; the compound has visible spectrum absorbance peaks at 413,435, 535, 578, 618 and 663 nanometers (195 270, 219 498, 14 052, 18 886,28 588, 86 733).

EXAMPLE 5 Production of "Chlorin NT2H2"

A solution was prepared by dissolving 100 mg Purpurin NT2 in 20 mgtetrahydrofuran and adding 2 drops of triethylamine; with stirring, anaddition 20 mg palladium on charcoal was made and the mixture whichresulted was hydrogenated at room temperature of about 22° for 5 hoursin a sloping manifold hydrogenator in which a slight positive pressureof hydrogen was maintained. The palladium on charcoal that was used wascomposed of 10 percent w/w of palladium and 90 percent w/w of charcoal.The palladium on charcoal was filtered from the colorless reactionmixture, and the filtrate was stirred vigorously while exposed to airuntil the solution turned brown, about 21/2 hours. The solvent was thenremoved in vacuo, and the residue was dissolved in the minimumdichloromethane containing 1 percent v/v of methanol and chromatographedon silica. A major blue band was collected; the solvent was removed; andthe crude product was dissolved in 5 ml dichloromethane containing 1percent v/v of methanol and recrystallized, yielding 75 mg brownmicroprisms which were identified by nuclear magnetic resonance asChlorin NT2H2, a compound having the formula of FIG. 8 of the drawingswhere R1 through R8 are ethyl, R9 is CO₂ CH₂ CH₃, and R10 through R13are hydrogen. Chlorin NT2H2 was found to have absorbance peaks in thevisible spectrum at 403, 500, 535, 558, 610 and 660 nanometers (114 650,23 532, 5 662, 4 246, 8 493, 39 455). The Chlorin NT2H2 zinc complex wasprepared by the method described in Example 4; it was found to haveabsorbance peaks in the visible spectrum at 408, 515, 545, 590 and 633nanometers (145 474, 9 858, 5 377, 15 832, 59 444).

The nickel complex of Chlorin NT2H2 was also prepared by the methoddescribed in Example 4, except that nickel acetate was substituted forthe zinc acetate. The nickel complex of Chlorin NT2H2 was found to haveabsorbance peaks in the visible spectrum at 405, 498, 533, 588 and 630nanometers (145 779, 11 034, 8 693, 19 392, 64 146).

The zinc and nickel complexes have the formula of FIG. 2 of the drawingswhere R1 through R8 are ethyl, R9 is CO₂ CH₂ CH₃ and R10 through R13 arehydrogen. M is Zn for the zinc complex and Ni for the nickel complex.

EXAMPLE 6 Production of Purpurin NT2 and Purpurin NT1

A solution of 100 mg meso-(-ethoxycarbonylvinyl)octaethyl porphyrin in20 ml glacial acetic acid was heated under reflux in air for 24 hours.The solution was allowed to stand at room temperature of about 22° untilit cooled; the solvent was removed in vacuo; and the residue wasdissolved in the minimum dichloromethane containing 1 percent v/v ofmethanol and chromatographed on silica. First and second major greenbands were collected; the solvent was removed from the first band; andthe crude product was dissolved in 4 ml dichloromethane containing 1percent v/v of methanol and recrystallized, yielding 40 mg "PurpurinNT1", a compound having the formula of FIG. 7 of the drawings where R1is ═CHCH₃ R2 through R8 are ethyl, R9 is CO₂ CH₂ CH₃ and R10 through R13are hydrogen. Purpurin NT1 was identified by nuclear magnetic resonance;it has absorbance peaks in the visible spectrum at wavelengths of 438,510, 540, 583, 653, and 715 nanometers (104 158, 9 450, 11, 130, 15 540,9 020, 42 629).

The solvent was also removed from the second green band, and the crudeproduct was dissolved in 4 ml dichloromethane containing 1 percent v/vof methanol and recrystallized, yielding 39 mg Purpurin NT2, which wasidentified by nuclear magnetic resonance.

Purpurin NT1 was hydrogenated by a procedure similar to that describedabove in Example 5, yielding, after work-up and chromatographicpurification as there described, 65 mg Chlorin NT2H2.

The procedure described in Example 3 has been used to produce otherpurpurins. Typical ones of the starting materials used and theintermediates and purpurins produced are set forth tabularly in Examples7, 8 and 9.

EXAMPLE 7

    ______________________________________                                                                    Formula                                           Compound                    of                                                ______________________________________                                        Starting material, Nickel meso-formyletio porphyrin I                                                     FIG. 6*                                           First Intermediate, Nickel meso-(-ethoxycarbonyl-                                                         FIG. 6*                                           vinyl)-etio porphyrin I                                                       Second intermediate, Meso-(-ethoxycarbonylvinyl)-etio                                                     FIG. 3*                                           porphyrin I                                                                   "Purpurin ET2"              FIG. 7*                                           ______________________________________                                         *Where: R1, R3, R5, and R7 are CH.sub.3, R2, R4, R6, and R8 are CH.sub.2      CH.sub.3.                                                                     In the starting material, R is CHO and M is Ni.                               In the first intermediate, R is CH═CHCO.sub.2 CH.sub.2 CH.sub.3 and M     is Ni.                                                                        In the second intermediate, R is CH═CHCO.sub.2 CH.sub.2 CH.sub.3.         In Purpurin ET2, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are     hydrogen.                                                                

The production of nickel meso-formyletio porphyrin I is disclosed in aJournal article by Johnson et al., J. Chem. Soc. (c) 1966, p. 794.

EXAMPLE 8

    ______________________________________                                                                    Formula                                           Compound                    of                                                ______________________________________                                        Starting Material, Nickel meso-formyl coproporphyrin                                                      FIG. 6**                                          I tetramethyl ester*                                                          First intermediate, Nickel meso-(-ethoxycarbonyl-                                                         FIG. 6**                                          vinyl)coproporphyrin I tetramethyl ester                                      Second intermediate, Meso-(-ethoxycarbonylvinyl)-                                                         FIG. 3**                                          coproporphyrin I tetramethyl ester                                            "Purpurin JP1"              FIG. 7**                                          ______________________________________                                         *Produced as subsequently described herein.                                   **Where: R1, R3, R5, and R7 are CH.sub.3 and R2, R4, R6 and R8 are            CH.sub.2 CH.sub.2 CO.sub.2 CH.sub.3.                                          In the starting material, R is CHO and M is Ni.                               In the first intermediate, R is CH═CHCO.sub.2 CH.sub.2 CH.sub.3 and M     is Ni.                                                                        In the second intermediate, R is CH═CHCO.sub.2 CH.sub.2 CH.sub.3.         In Purpurin JP1, R9 is CO.sub.2 CH.sub.2 CH.sub.3 and R10 through R13 are     hydrogen.                                                                

The nickel meso-formyl coproporphyrin I tetramethyl ester startingmaterial used in the procedure of Example F was produced from acommercially available material, coproporphyrin I tetramethyl ester(formula of FIG. 3 of the attached drawings); nickel coproporphyrin Itetramethyl ester was produced therefrom (formula of FIG. 6 where M isZn). In both cases, R1, R3, R5 and R7 are CH₃ and R2, R4, R6 and R8 areCH₂ CH₂ CO₂ CH₃.

The Ni Coproporphyrin I Tetramethyl ester was prepared from a solutionof 100 mg coproporphyrin I tetramethyl ester in a mixed solvent composedof 50 ml dichloromethane and 5 ml methanol and 100 mg nickel acetate. Amixture which was prepared by adding the nickel acetate to the solutionwas refluxed for about 12 hours until the electronic spectrum of thereaction mixture indicated that chelation was complete. The reactionmixture was then concentrated to 7 ml and allowed to cool to roomtemperature of about 22°. Product which precipitated was recovered byfiltration, dissolved in a mixed solvent composed of 5 mldichloromethane and 2 ml methanol, and recrystallized, yielding 98 mg Nicoproporphyrin I tetramethyl ester. The compound showed absorbance peaksin the visible spectrum at 392, 515 and 552 nanometers; the relativeintensities at these peaks were 20.19, 1 and 2.56, respectively.

The Nickel-meso-formyl coproporphyrin I tetramethyl ester was preparedfrom 2.8 ml freshly distilled phosphorus oxychloride, 2 ml dry dimethylformamide, a solution of 100 mg nickel-coproporphyrin I tetramethylester in 75 ml dry 1,2-dichloroethane and 75 ml saturated aqueous sodiumacetate. The dimethyl formamide was cooled on an ice bath, and thephosphorus oxychloride was added thereto dropwise. The solution whichresulted was allowed to stand at room temperature for 30 minutes, andwas then warmed to 50°. The nickel-coproporphyrin I tetramethyl estersolution was then added dropwise, with stirring, over 30 minutes to thephosphorus oxychloride solution. The reaction mixture was maintained atabout 50°, with stirring, for an additional 2 hours, during which time achange in color from red to green was observed. The sodium acetatesolution was then added to the reaction mixture, and stirring wascontinued for an additional 2 hours. The organic and the aqueous phaseswere then separated, and the aqueous phase was extracted withdichloromethane. The organic phase and the dichloromethane extract werethen combined, and evaporated to dryness. The solid which remained wasrecrystallized from a solvent composed of equal parts by volume ofdichloromethane and methanol, yielding 86 mg red microcrystals whichwere identified by nuclear magnetic resonance asnickel-mesoformylcoproporphyrin I tetramethyl ester. Absorbance peakswere found in the visible spectrum at 400, 420, 558 and 645 nanometers,with relative intensities of 10.10, 8.69, 1.02 and 1, respectively.

EXAMPLE 9

    ______________________________________                                                                    Formula                                           Compound                    of                                                ______________________________________                                        Starting Material, Nickel meso-formyloctaethyl-                                                           FIG. 6*                                           porphyrin                                                                     First intermediate, Nickel meso-(-methoxycarbonyl-                                                        FIG. 6*                                           vinyl)octaethylporphyrin                                                      Second intermediate, Meso-(-methoxycarbonyl-                                                              FIG. 3*                                           vinyl)octaethylporphyrin                                                      "Purpurin GG2"              FIG. 7*                                           ______________________________________                                         *Where: R1 through R8 are CH.sub.2 CH.sub.3.                                  In the starting material, R is CHO and M is Ni.                               In the first intermediate, R is CH═CHCO.sub.2 CH.sub.3 and M is Ni.       In the second intermediate, R is CH═CHCO.sub.2 CH.sub.3.                  In Purpurin GG2, R9 is CO.sub.2 CH.sub.3 and R10 through R13 are hydrogen                                                                              

The procedure of Example 5 has been used to hydrogenate Purpurin ET2 andPurpurin JP1, producing Chlorin ET2H2 and Chlorin JP1H2, respectively,where the isocyclic ring (to which the R9 substituent is attached issaturated. The chlorins had the same substituents as the startingpurpurins, but the structure of FIG. 8 instead of that of FIG. 7.

The procedure of Example 6 has been used to produce other zinc andnickel complexes. The purpurin and chlorin starting materials, the zincor nickel compound used, and the complexes produced are set forth below:

    ______________________________________                                        Starting       Zinc or nickel                                                                            Complex                                            Purpurin or Chlorin                                                                          Compound    Produced                                           ______________________________________                                        Purpurin ET2   Zinc acetate                                                                              Zn                                                 Purpurin ET2   Nickel acetate                                                                            Ni                                                 Purpurin GG2   Zinc acetate                                                                              Zn                                                 Purpurin GG2   Nickel acetate                                                                            Ni                                                 Chlorin ET2H2  Zinc acetate                                                                              Zn                                                 Chlorin ET2H2  Nickel acetate                                                                            Ni                                                 ______________________________________                                    

Additional peak absorbance data (visible spectrum, wavelengths innanometers) is given below.

    ______________________________________                                        Compound    Wavelengths (relative intensities)                                ______________________________________                                        Purpurin ET2                                                                              406(16.69), 424(15.26), 502(1.36), 531(1),                                    566(1.5), 695(3.47)                                               Chlorin ET2H2                                                                             400(70.16), 498(5.53), 530(1.29), 555(1),                                     606(1.91), 662(20.82)                                             Zn Chlorin ET2H2                                                                          401(20.36), 530(1), 568(1.18), 630(3.20)                          Purpurin ET2                                                                              434(16.44), 530(1), 576(1.31) 61.2(1.77),                                     660(5.0)                                                          Ni Purpurin ET2                                                                           434(5.14), 657(1)                                                 Ni Chlorin ET2H2                                                                          404(11.70), 497(1), 622(4.41)                                     Purpurin JP1                                                                              409(22.41), 504(1.67), 541(1.21) 576(1.08),                                   647(1), 691(3.79)                                                 Chlorin JP1H2                                                                             401(14.53), 650(1)                                                Purpurin GG2                                                                              406(12.94), 427(19.18), 500(1), 526(1),                                       565(1.89), 637(0.76), 695(5.25)                                   Zn Purpurin GG2                                                                           436(8.33), 616(1), 661(3.43)                                      Ni Purpurin GG2                                                                           427(4.20), 648(1)                                                 ______________________________________                                    

In vitro and in vivo testing of purpurins and chlorins produced asdescribed in Examples 3 through 9 was also carried out. For the in vitrotesting, the compounds were dissolved in dimethyl sulfoxide or in asolvent that is commercially available under the trade designationPROTOSOLV, and diluted with phosphate buffer saline to a concentrationof 0.010 mg per ml. The tests were conducted on FANFT(N-[4-(5-nitro-2furyl)2-thiazolyl] formamide) induced rat bladder tumorcells. Two tests were conducted, uptake and toxicity.

The uptake test involved incubating the FANFT inducted rat bladder tumorcells with a solution of a purpurin or with a solution of a chlorin at aconcentration of 0.010 mg per ml for one hour, temperature 37°, followedby removal of the incubation media, three washes of the cells withphosphate buffered saline, and extracting and quantitating of thepurpurin or chlorin retained by the cells. The procedure as used ininvestigating the use of HpD in rat tumor cells is described in detailin a journal article by Garbo et al., Analytical Biochemistry, Vol. 151(No. 1), pp. 70-81, 1985.

The toxicity test involved the incubation and washing steps of theuptake test, followed by illumination of the cells with red light of awavelength greater than 590 nanometers. Cell survival was thendetermined by Trypan Blue exclusion, a technique described in a journalarticle by Schneck, R., Arch. Path. (Lab. Med.), 35, p. 857, 1943.

The uptake test was positive for Purpurin NT2 and for Chlorin NT2H2. Theresults of the toxicity test are given in the following table, togetherwith the results of toxicity testing of HpD, of phosphate buffer salineand of the solvent system in which the purpurin or chlorin wasdissolved.

    ______________________________________                                        Test Solution    Average Viability                                            ______________________________________                                        Purpurin NT2     46                                                           Chlorin NT2H2    51                                                           HpD              42                                                           Phosphate buffer saline                                                                        93                                                           Mixed solvent    96                                                           ______________________________________                                    

The in vivo testing was conducted on male Fisher 344 rats weighing 135to 150 g in whom the transplantable FANFT(N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide tumor system had beenimplanted. (Use of this system is reported by Selman, S. H., et al.,Cancer Research, pp. 1924-1927, May, 1984.) Two tumors were implantedinto the subcutaneous tissue of the abdominal wall of each test animal;when the testing was carried out, each tumor was about 1 cm in diameter.

The purpurins and chlorins tested were dissolved in a commerciallyavailable non-ionic solubilizer and emulsifier obtained by reactingethylene oxide with castor oil in a ratio of 35 moles of ethylene oxideper mole of castor oil, diluting the resulting solution with1,2-propanediol, and producing an emulsion with the resulting solutionand 0.9 percent w/w aqueous sodium chloride solution. The specificnon-ionic solubilizer used is available from BASF under the designationCREMOPHOR EL; it is composed of fatty acid esters of polyglycols,glycerol polyglycols, polyethylene glycols and ethoxylated glycerol. Thetest solutions were prepared from 50 mg purpurin or chlorin, 1 or 2 mlwarm solubilizer (enough to dissolve the test compound), enough1,2-propanediol to make a solution of the purpurin or chlorin in a mixeddiol/solubilizer solvent containing 32.9 percent w/w solubilizer;finally, enough 0.9 percent w/w aqueous sodium chloride was added tomake 10 ml test solution so that the final concentration of the purpurinor chlorin in the test solution was 5 mg per ml. Each test solution wasmade, with mechanical shaking and stirring, by dissolving the purpurinor chlorin in the solubilizer, diluting the resulting solution with theindicated amount of 1,2-propanediol, and adding the sodium chloridesolution to the diluted solution. A control solution was also preparedfor use with each test solution. The control was identical with the testsolution except that it contained no purpurin or chlorin. The testsolutions were prepared in air, but it is believed that a nitrogenatmosphere would be advantageous because it would minimize the chance ofa reaction with oxygen.

The testing involved injecting each rat with a solution of the purpurinor chlorin under test, dosage 4 mg purpurin or chlorin per kg of bodyweight or 10 mg purpurin or chlorin per kg of body weight or with thesame volume of the appropriate control, irradiating one of the twotumors with light for 30 minutes, sacrificing the animals, and examiningthe tumors. The injections were made via the dorsal tail vein. Theirradiation of one of the tumors occurred twenty four hours after eachrat was injected while the other of the two tumors was shielded by anopaque box.

Tumor temperature and body core temperature were monitored, usingthermistors, one placed into the tumor and one placed intrarectally.Tumor temperature was kept within 2° of body core temperature bydirecting a jet of cool air over the tumor.

The light source was a slide project that had a 500 watt bulb fittedwith a red filter which is available from Corning Glass Works under thedesignation 2418. The light was reflected 90 by a silvered mirror, andwas focused onto the tumor with a secondary condensing lens. The lightintensity on the tumor was monitored, using a photometer/radiometer thatis available from United Detector Technology under the designation "UDT#351", and was maintained at 200 mw per cm².

Six rats were injected with the purpurin or chlorin test solution andtwo were injected with the appropriate control solution.

Four hours after the irradiation, three of the rats that had beeninjected with the test solution and one of the rats that had beeninjected with the control were sacrificed by an intracardiac injectionof saturated aqueous potassium chloride solution. Twenty four hoursafter the irradiation, another three of the rats that had been injectedwith the test solution and the other rat that had been injected with thecontrol were sacrificed in the same way. During the testing, the ratswere under barbiturate anesthesia (65 mg per kg body weight).

The tumors were then excised, placed in 10 percent w/wphosphate-buffered formalin and cut into three sections perpendicular totheir long axis. The tumors were then embedded in paraffin and cut intosections five microns in width. The sections were stained withhematoxylin and eosin.

Histologic examination of the stained sections revealed approximatelycomparable areas of hemorrhage and tumor cell necrosis in specimensremoved four hours after irradiation from animals that had been injectedwith Purpurin NT2, with Purpurin GG2, and with Purpurin ET2. However,tumor cells which appeared to be viable were observed. Only minorhemorrhage and tumor cell necrosis were observed in specimens removedfour hours after irradiation from animals that had been injected withPurpurin JP1 but much greater hemorrhage and necrosis were observed inspecimens that had been injected with Purpurin ZnET2 and even more inspecimens that had been injected with Chlorin SnET2H2. Tumor necrosiswas extensive in specimens removed twenty four hours after irradiationfrom animals that had been injected with Purpurin NT2, with PurpurinGG2, with Purpurin ET2, with Purpurin JP1, with Purpurin ZnET2 and withChlorin SnET2H2; no viable tumor cell was observed in specimens fromanimals that had been injected with Purpurin ZnET2 and Chlorin SnET2H2,while a few were observed in specimens from animals that had beeninjected with Purpurin NT2 and with Purpurin ET2, and more were observedin specimens from animals that had been injected with Purpurin GG2 andwith Purpurin JP2. No change in the tumors was observed in the specimensthat were removed from animals that had been injected with the controlsolution. Tumor necrosis was complete in specimens removed from animalsthat had been injected with purpurin NT1 both four hours afterirradiation and twenty four hours after irradiation. However, theirradiation was found to have caused extensive liver damage to some ofthe animals. The liver damage is believed to have occurred because therewas residual Purpurin NT1 in the liver which was unintentionallyirradiated. The in vivo testing, however, indicated that Purpurin NT1 ishighly effective when properly used.

The in vivo test procedure described above has also been used toevaluate solutions in which the Purpurin NT2, Purpurin GG2, PurpurinNT1, Purpurin JP1, Purpurin ET2, Purpurin ZnET2 and Chlorin SnET2H2 werereplaced by Chlorin NT2H2 and by Chlorin ET2H2. Histologic examinationof the stained sections from rats into which the Chlorin NT2H2 and ET2H2solutions had been injected indicated that these chlorins weresubstantially equivalent in this test and were similar to Purpurin NT2,to Purpurin GG2 and to Purpurin ET2, the only difference observed beingthat hemorrhage within the tumors was less pronounced with the chlorins.

It will be appreciated from the results reported above in Example 6 thatthe cyclization step of Example 1 by which Purpurin II and Purpurin IIIare produced could be carried out in air, rather than in nitrogen, andthat the reaction product would then be a mixture of Purpurin II,Purpurin III, and the following purpurins:

    ______________________________________                                        Compound  Structure (referring to attached drawings)                          ______________________________________                                        Purpurin XXI                                                                            FIG. 9:  R1 is ═CHCH.sub.3, R2-R5, R7 and R8                                       are CH.sub.2 CH.sub.3 R9, and is CO.sub.2 CH.sub.2                            CH.sub.3                                                   Purpurin XXII                                                                           FIG. 13: R1 is ═CHCH.sub.3, R2-R6 and R8 are                                       CH.sub.2 CH.sub.3, and R9 is CO.sub.2 CH.sub.2             ______________________________________                                                           CH.sub.3                                               

In fact, cyclization in air can be used to produce purpurins, and metalcomplexes can be produced from those purpurins by the method of Example4, with or without the modifications thereof subsequently describedherein, where the purpurins and complexes have the structures of FIGS.10-12, of FIGS. 14-18, of FIGS. 29-33, of FIGS. 34-38, of FIGS. 44-48and of FIGS. 54-58. In all cases, a mixture of products will beproduced, some in which R1 is saturated and some in which R1 is abivalent aliphatic hydrocarbon radical having from 2 to 4 carbon atomswherein both of the valences of the radical are attached to the samecarbon atom thereof and to a carbon atom of the purpurin or metalcomplex. Furthermore, after cleavage of the exocyclic ring of FIGS.9-18, oxidation in air can be used, in the purpurins and complexes ofFIGS. 29-33 and of FIGS. 44-48 to convert R2 to a bivalent aliphatichydrocarbon radical having from 2 to 4 carbon atoms wherein both of thevalences of the radical are attached to the same carbon atom thereof andto a carbon atom of the purpurin or metal complex. In general, purpurinscan be converted to the corresponding chlorins by the hydrogenationmethod described in Example 5; chlorins can be converted to thecorresponding purpurins by oxidation; and chlorin metal complexes can beproduced from chlorins by the method of Example 4, with or without themodifications thereof subsequently described herein. However,hydrogenation of Purpurin NT1 (where there was a double bond between theR1 substituent and a carbon of the purpurin), as described in Example 6,produced Chlorin NT2H2 (where both the double bond of the exocyclic ringand that of R1 were saturated). It will be appreciated that the R1double bond forms in the Example 6 procedure because a hydroxyl group isintroduced into the molecule and, at the temperature of reflux, theelements of water are eliminated to form the double bond. Elimination ofthe elements of water can be prevented in the Example 6 procedure, or,more generally, whenever R1 is to be bivalent, by cyclizing at a lowertemperature; the resulting purpurin can then be hydrogenated to thecorresponding chlorin and the double bond with R1 can be formed byheating.

The method of Example 4, supra, can be used to produce metal complexesof other purpurins and of various chlorins. Specifically, an equivalentamount of another purpurin or of a chlorin can be substituted for thePurpurin NT2, or copper acetate, nickel acetate, cobalt acetate, silveracetate, palladium acetate, or platinum acetate can be substituted forthe zinc acetate, or both substitutions can be made. In this manner,purpurin metal complexes having the formulas of FIGS. 54-58 where M isone of the metals named above in this paragraph can be produced frompurpurins having the formulas of FIGS. 34-38; chlorin metal complexeshaving the formulas of FIGS. 49-53 where M has the same meaning can beproduced from chlorins having the formulas of FIGS. 39-43; purpurinmetal complexes having the formulas of FIGS. 44-48 where M had theindicated meaning can be produced from purpurins having the formulas ofFIGS. 29-33; metal complexes of purpurins having the formulas of FIGS.9-18 can be produced; metal complexes having the structure of FIG. 1 canbe produced from purpurins having the structure of FIG. 7; and metalcomplexes having the structure of FIG. 2 can be produced from chlorinshaving the structure of FIG. 8. Other complexes can be produced by themethod of Example 4 from salts containing cations other than acetate,and producing complexes which have the structures of the Figs. to whichreference is made above in this paragraph, but where M does notrepresent merely a metal anion. Examples of salts that can besubstituted for zinc acetate in the Example 4 procedure are given below,together with the identify of M in the foregoing Figs.:

    ______________________________________                                        Salt                Identity of M                                             ______________________________________                                        FeCl.sub.3          Fe(Cl)                                                    MnCl.sub.4          Mn(Cl)                                                    InCl.sub.3          In(Cl)                                                    VCl.sub.4 *         V(O)                                                      Tl(CF.sub.3 CO.sub.2).sub.3                                                                       Tl(OAc)(H.sub.2 O)                                        SnCl.sub.2          Sn(OH).sub.2                                              [Rh(CO).sub.2 Cl].sub.2                                                                           Rh(Cl)(H.sub.2 O)                                         ______________________________________                                         *Using phenol as the solvent instead of glacial acetic acid.             

The procedure of Example 4 can also be modified by substituting phenolfor glacial acetic acid and metal chelates of pentane, 2,4-dione forzinc acetate to produce complexes of any of the foregoing purpurins andchlorins. Metals that can be so reacted (as pentane, 2,4-dione chelates)and the identity of M in the complex that is produced are set forth inthe following table:

    ______________________________________                                        Metal     Identity of M                                                                             Metal     Identity of M                                 ______________________________________                                        Al        Al(acac)*   Th        Th(acac).sub.2                                Sc        Sc(acac)    U         U(acac).sub.2                                 Ga        Ga(acac)    La        La(acac).sub.2                                In        In(acac)    Ce        Ce(acac)                                      Mo        Mo(acac)    Nd        Nd(acac)                                      Ti        Ti(acac).sub.2                                                                            Sm        Sm(acac)                                      Zr        Zr(acac).sub.2                                                                            Gd        Gd(acac)                                      Hf        Hf(acac).sub.2                                                                            Tb        Tb(acac)                                      Eu        Eu(acac)    Dy        Dy(acac)                                      Pr        Pr(acac)    Ho        Ho(acac)                                      Yb        Yb(acac)    Er        Er(acac)                                      Y         Y(acac)     Tm        Tm(acac)                                      Lu        Lu(acac)                                                            ______________________________________                                         *The pentane, 2,4dione portion of a chelate thereof with a metal.        

Complexes of any of the foregoing purpurins and chlorins can also beproduced by the procedure of Example 4, substituting dimethylformamidefor glacial acetic acid and CrCl₂ for zinc acetate. Metal complexformation occurs at higher temperatures when dimethylformamide is used,because of its higher boiling temperature. M in the complexes is Cr(OH).

Similarly, complexes of the foregoing purpurins and chlorins can beproduced by the procedure of Example 4, substituting pyridine forglacial acetic acid and PbCl₂ for zinc acetate. M in the complexes isPb.

The method of Example 4 and the modifications thereof described abovecan be used to produce purpurin complexes having the structures of FIGS.44-48 from purpurins having the structures of FIGS. 29-33; to producechlorin complexes having the structures of FIGS. 49-53 from chlorinshaving the structures of FIGS. 39-43; to produce purpurin complexeshaving the structures of FIGS. 54-58 from purpurins having thestructures of FIGS. 34-38; to produce purpurin complexes from purpurinshaving the structure of FIG. 7; and to produce chlorin complexes of FIG.2 from chlorins having the structure of FIG. 8.

The following example illustrates the production of Chlorin I, acompound having the structure of FIG. 19 where R1-R5, R7 and R8 are CH₂CH₃, Chlorin II, a compound having the structure of FIG. 24 where R1-R5,R7 and R8 are CH₂ CH₃ and R14 is CH₃, and Purpurin XXIII, a compoundhaving the structure of FIG. 29 where R1-R5, R7 and R8 are CH₂ CH₃, R10and R12 are H, R14 is CH₃ and R15 is CO₂ CH₂ CH₃.

EXAMPLE 10 Production of Chlorin I and Chlorin II

A 5 percent w/w solution of 100 mg Purpurin IV in dichloromethanecontaining 25 percent v/v methanol is caused to react by vigorousstirring in air while illuminated with visible light. The reaction iscontinued, with periodic monitoring by visible light spectroscopy, untilthe spectrum indicates that no Purpurin IV remains in the solvent. Thedichloromethane/methanol solvent is then evaporated; a 4 ml portion of a5 percent w/w solution of sodium methoxide in methanol is mixed with theresidue and refluxed for 2 hours; and the solution is cooled. A 5 mlportion of water is then mixed with the reaction product; an organiclayer which forms is separated from the aqueous layer and treated invacuo to remove solvent; and the crude product which remains isdissolved in about 3 ml dichloromethane containing 1 percent v/vmethanol and chromatographed on silica gel. The CHO group of Chlorin Iis then reduced to CH₃, for example by the method described above asExample 5, producing Chlorin II.

Production of Purpurin XXIII

A solution of 60 mg sodium borohydride in 10 ml methanol is addeddropwise to a solution of 200 mg Chlorin II in 5 ml dichloromethane; theresulting solution is stirred at room temperature of about 22° for 2hours, and is poured into 100 ml water. The organic phase is separatedfrom the aqueous phase; the solvent is removed from the organic phase;and the solvent is evaporated. The residue is dissolved in 50 mlchloroform containing 25 percent v/v methanol; a 10 mg addition ofp-toluene sulfonic acid is made; and the reaction mixture is refluxedfor 6 hours. Water is then added to the reaction mixture; the organiclayer is collected; and the solvent is removed by evaporation. Theresidue is dissolved in 5 ml dichloromethane containing 2 percent v/vmethanol; the resulting solution is chromatographed on silica gel; andPurpurin XXIII is recovered by evaporating the solvent from thechromatographed solution.

The procedure of Example 10 can also be used to produce other chlorinsand other purpurins. For example, Purpurin XI can be substituted forPurpurin IV and Chlorin III, Chlorin IV and Purpurin XXIV can then beproduced by the method of Example 10; these compounds are identified inthe following table:

    ______________________________________                                        Compound    Structure (referring to attached drawings)                        ______________________________________                                        Chlorin III FIG. 20: R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                    Chlorin IV  FIG. 25: R1-R3 and R5-R8 are CH.sub.2 CH.sub.3                                and R14 is CH.sub.3                                               Purpurin XXIV                                                                             FIG. 30: R1-R3 and R5-R8 are CH.sub.2 CH.sub.3,                               R10 and R11 are HR14 is CH.sub.3, and                                         R15 is CO.sub.2 CH.sub.2 CH.sub.3                                 ______________________________________                                    

Similarly, Purpurin XVI can be substituted for Purpurin IV and ChlorinV, Chlorin VI and Purpurin XXV can then be produced by the method ofExample 10; these compounds are identified in the following table:

    ______________________________________                                        Compound  Structure (referring to attached drawings)                          ______________________________________                                        Chlorin V FIG. 21: R1-R7 are CH.sub.2 CH.sub.3                                Chlorin VI                                                                              FIG. 26: R1-R7 are CH.sub.2 CH.sub.3 and R14 is CH.sub.3            Purpurin XXV                                                                            FIG. 31: R1-R7 are CH.sub.2 CH.sub.3, R11 and R12                             are H, R14 is CH.sub.3, and                                                   R15 is CO.sub.2 CH.sub.2 CH.sub.3                                   ______________________________________                                    

In a like manner, Purpurin XIX can be substituted for Purpurin IV andChlorin VII, Chlorin VIII and Purpurin XXVI can then be produced by themethod of Example 10; these compounds are identified in the followingtable:

    ______________________________________                                        Compound  Structure (referring to attached drawings)                          ______________________________________                                        Chlorin VII                                                                             FIG. 22: R1-R4 and R6-R8 are Ch.sub.2 CH.sub.3                      Chlorin VIII                                                                            FIG. 27: R1-R4 and R6-R8 are CH.sub.2 CH.sub.3, and                                    and R14 is CH.sub.3                                        Purpurin XXVI                                                                           FIG. 32: R1-R4 and R6-R8 are CH.sub.2 CH.sub.3, R10                                    and R11 are H, R14 is CH.sub.3, and R15                                       is CO.sub.2 CH.sub.2 CH.sub.3                              ______________________________________                                    

Finally, Purpurin VII can be substituted for Purpurin IV and Chlorin IX,Chlorin X and Purpurin XXVII can then be produced by the method ofExample 10; these compounds are identified in the following table:

    ______________________________________                                        Compound    Structure (referring to attached drawings)                        ______________________________________                                        Chlorin IX  FIG. 23: R1-R6 and R8 are CH.sub.2 CH.sub.3                       Chlorin X   FIG. 28: R1-R6 and R8 are CH.sub.2 CH.sub.3, and                                       R14 is CH.sub.3                                          Purpurin XXVII                                                                            FIG. 33: R1-R6 and R8 are CH.sub.2 CH.sub.3, R10                                       and R12 are H, R14 is CH.sub.3, and                                           R15 is CO.sub.2 CH.sub.2 CH.sub.3                        ______________________________________                                    

It will be appreciated that the purpurins that can be produced by themethods of the foregoing examples, and the chlorins that can be producedfrom those purpurins by the hydrogenation method of Example 5, haveseven substituents which are present in the porphyrin starting materialsfrom which the purpurins are produced. These are seven of the eightR1-R8 substituents, all but R6 in the purpurins of FIG. 34, all but R4in the purpurins of FIG. 35, etc. The identifies of these substituentsdepend on the identities of their pyrrole-, dipyrromethane-, andporphyrin precursors. At least as initially produced, each purpurin alsohas an R9 substituent, an R15 substituent, or both; as is explainedabove, the identities of these substituents are determined by theidentities of their porphyrin precursors. In addition, the purpurins ofFIGS. 29-33 have the potential for substitution at R14, which is formylwhen the purpurins are first produced, as described above, and can bereduced to methyl, as also described above, or to any other desiredsubstituent by the reactions about to be described to which a formylgroup introduced as R10, R11, R12, R13 or R16 can be subjected. Finally,each purpurin has additional sites for potential substitution, aplurality of R10, R11, R12, R13 and R16 (see FIGS. 34-38) substitutionsbeing possible, which ones depending on the position of an exocyclicring to which R15 and R16 are attached; as the purpurins are produced,there is hydrogen in each of these positions. A formyl group can beintroduced by reaction with the Vilsmier reagent as the lowest of R10,R11 and R12, as R13 and as R16 (in the compounds of FIGS. 34-38), or theunsaturated exocyclic ring or rings can be saturated by hydrogenation(see procedure of Example 5, supra) and the formyl group can then beintroduced as the lowest of R10, R11 and R12. The formyl group, afterseparation of isomers, if necessary, can be reduced to CH₃, or can bereduced to CH₂ OH or converted to an oxime group, which can then beconverted to a cyano group, which, in turn, can be converted to anamide. The formyl group can also be reacted with Wittig reagents to givealkyl, alkenyl or carboxy side chains or to introduce the previouslyidentified substituents which have an amine or an alcoholic OH functionas the lowest of R10, R11 and R12, as R13 or as R16. After the desiredgroup has been introduced as the lowest of R10, R11 and R12, as R13, asR16, or as some combination, the purpurin can be reacted in the same wayto introduce a desired group as R11, if present. Finally, the chemistrycan be used to introduce a desired group as R12, is present.

As has been indicated above, the instant invention, in one aspect, is apurpurin, a chlorin or a metal complex which has a structure that hasbeen enriched in an atom that can be detected by nuclear magneticresonance. Such purpurins are produced by repeating the procedure ofExample 1, but producing Pyrrole I from a saturated aqueous solutioncontaining one gram equivalent of sodium nitrite enriched in N-15, a 5percent w/w solution in glacial acetic acid containing one gramequivalent of benzyl propionylacetate, a suspension in glacial aceticacid of one gram equivalent of ethyl acetoacetate and four gramequivalents of zinc dust, and producing Pyrrole III from a saturatedaqueous solution containing one gram equivalent of sodium nitriteenriched in N-15, a 5 percent w/w solution in glacial acetic acidcontaining one gram equivalent of benzyl propionylacetate, a suspensionin glacial acetic acid of one gram equivalent of 2,4-pentanedione andfour gram equivalents of zinc dust. A 10 percent enrichment of thesodium nitrite in N-15 is adequate to produce Purpurin I, Purpurin II,Purpurin III, Purpurin IV and Purpurin V enriched in N-15 to such anextent that, after they have been administered intravenously asdescribed above, their location in the patient to whom they wereadministered can be monitored by nuclear magnetic resonance.

Purpurins that can be detected by nuclear magnetic resonance are alsoproduced by repeating Example 1, but producing Porphyrin Complex VIIfrom a solution in 50 ml xylene of 506 mg Porphyrin Complex V and 1.024g (carbethoxymethylene)triphenyl-phosphorane in which thecarbethoxymethylene moiety is enriched in C-13. A 10 percent enrichmentof the carbethoxymethylene moiety in C-13 is adequate to producePurpurin I, Purpurin II, Purpurin III, Purpurin IV and Purpurin Venriched in C-13 to such an extent that, after they have beenadministered intravenously as described above, their location in thepatient in whom they were administered can be monitored by nuclearmagnetic resonance.

As has also been indicated above, the instant invention, in anotheraspect, is a purpurin, a chlorin or a metal complex which has astructure that has been enriched in an atom that is radioactive to suchan extent that its presence can be detected by an instrument thatmeasures the level of ionizing radiation. Such purpurins are produced byrepeating the procedure of Example 1, but producing Porphyrin ComplexVII from a solution in 50 ml xylene of 506 mg Porphyrin Complex V and1.024 g (carbethoxymethylene)triphenylphosphorane in which thecarbethoxymethylene moiety is enriched in C-14. A 10 percent enrichmentof the carbethoxymethylene moiety in C-14 is adequate to producePurpurin I, Purpurin II, Purpurin III, Purpurin IV and Purpurin Venriched in C-14 to such an extent that, after they have beenadministered intravenously as described above, their location in thepatient to whom they were administered can be monitored by an instrumentwhich measures the level of ionizing radiation. C-14 has an extremelylong half life; it will be appreciated, therefore, that a purpurin,chlorin or complex that has a structure which is enriched in C-14 shouldnot be administered to a human, but that such a compound can beadministered to a laboratory animal and that monitoring its location inthe body of the laboratory animal can then provide extremely valuableinformation which has application in the treatment of humans.

Purpurins, chlorins and complexes having structures which are enrichedin an atom that emits ionizing radiation and which are suitable foradministration to humans can also be produced. For example, any of thepurpurins according to the invention where at least one of R10 throughR14 is hydrogen can be reacted in sunlight with elemental I-131 or with¹³¹ ICl, and chlorins and complexes can be produced as described abovefrom the iodinated purpurin which is produced. Further, purpurin andchlorin complexes can be produced as described above from a gallium ormolybdenum chelate of pentane, 2,4-dione where the gallium is Ga-67, orthe molybdenum is Mo-99. Mo-99 becomes Tc-99m, which, like Ga-67 andI-131, is physiologically acceptable for use as a tracer in humanpatients. Accordingly, such compounds containing I-131 and the Ga-67 andTc-99m complexes are suitable for administration to humans.

It will be appreciated that Purpurins I through V produced as describedabove, and enriched in N-15, in C-13, in C-14, or in I-131 can be usedas also described above to produce other purpurins which are so enrichedand that the methods of the examples hereof can be varied as describedabove to produce purpurins having the structures of FIGS. 7, 9-18, 29-33and 34-38 which are enriched in N-15, in C-13 or in C-14, and whereineach of R1 through R8 has the meaning set forth above. Similarly, themethod described above can be used to produce purpurins that are soenriched where each of R9 through R16 has the meaning set forth above.Likewise, chlorins and purpurin metal complexes can be produced fromthose purpurins as described above, and metal complexes can be producedfrom the chlorins as so described.

As is indicated above, there are indications that the purpurins,chlorins and metal complexes are X ray sensitizers which increase thetherapeutic ratio of X rays. Accordingly, in one aspect, the instantinvention involves administering, for example, as described above, apurpurin having the structure of one of FIG. 7, 9-18 and 29-38, acorresponding chlorin or a chlorin or purpurin metal complex and, afterthe purpurin, chlorin or complex has localized, treating the affectedregion with X rays or other ionizing radiation.

As is also indicated above, the purpurins, chlorins and complexes can beadministered topically, for example as dilute, e.g., 1 percent w/wsolutions in DMSO or ethanol to non-malignant lesions, e.g., of thevagina or bladder, or to such cutaneous lesions as are involved inpsoriasis, followed by illumination of the area involved with light of awavelength at which the purpurin, chlorin or complex has an absorbancepeak. The purpurin or the like solution should be applied only to thelesions to prevent damage to healthy tissue adjacent the lesions.Illumination of the lesions, for example, for from 15 to 30 minutes thencompletes the treatment. It is to be understood, however, thatpurpurins, chlorins and complexes according to the invention can also beadministered systemically in the treatment of non-malignant lesions.

The reaction of a monoclonal antibody with Purpurin II is described inExample 2. The monoclonal antibody, when it is one which localizes intumors, can enhance the ability of the purpurin, or of a chlorin orcomplex produced therefrom, to localize in tumors, as discussed above,However, the monoclonal antibody can also be of a different type, forexample one which localizes in a particular kind of lymphocyte, in aleukemia cell, in a lymphoma cell, or the like; a product of thereaction of Purpurin II or the like with such an antibody whichlocalizes in a particular kind of lymphocyte can be used to modulatelymphocyte populations in the treatment of immune diseases, e.g.,arthritis, or to re-establish a lymphocyte balance in transplantpatients. Some of the blood is removed from the patient's body, and sucha purpurin, chlorin or complex according to the invention, i.e., onewhere at least one of the substituents is a monoclonal antibody directedagainst the lymphocyte or lymphocytes present in excess, is mixed withthe blood in a suitable amount; after the purpurin or the like localizesin the lymphocyte or lymphocytes present in excess, the blood is exposedto light of a wave length at which the purpurin or the like has anabsorbance peak, destroying the lymphocyte or lymphocytes wherelocalization had occurred. The blood is then returned to the patient'sbody. This technique can be carried out repeatedly as required tomodulate lymphocyte populations in treating immune diseases andtransplant patients who develop the lymphocyte imbalance that isassociated with the rejection of a transplanted organ. Since thetreatment is entirely outside the patient's body, there is noopportunity for the development of a natural resistance to the treatmentwhich is characteristic or prior attempts to modulate lymphocytepopulations. Purpurins or the like according to the invention where oneof the substituents is an antibody against leukemia cells or againstlymphoma cells can be used in a similar manner in the treatment ofleukemia and lymphoma.

It will be appreciated that purpurins and chlorins according to theinvention where R10 through R13 and R16 are hydrogen are preferred,other factors being equal, because the production of the compounds withother groups in these positions is complicated, time consuming andexpensive. R9 and R15 in chlorins and purpurin and chlorin complexesaccording to the invention are preferably CO₂ R' where R' is a primaryor secondary alkyl group having from 1 to 4 carbon atoms, other factorsbeing equal, because these groups are present at the end of the ringclosure reaction which produces the purpurins (see Examples 1 and 3); R9and R15 in purpurins according to the invention are preferably CO₂ R'where R' is a primary or secondary alkyl group having from 2 to 4 carbonatoms, other factors being equal, and for the same reason. However, theesters of these R9 and R15 substituents can be reduced to formyl groupsand reacted as discussed above to introduce any of the R1 to R8, R10 toR14 and R16 substituents.

The production of purpurin solutions in the specific non-ionicsolubilizer that is available under the designation CREMOPHOR EL, andthe production of emulsions of such solutions with 1,2-propanediol andsaline solution is described above, as is the use of such solutions todetect and treat tumors. It will be appreciated that purpurins, chlorinsand their metal complexes can be dissolved in other non-ionicsolubilizers and that the solutions can be used to produced emulsionsthat can be administrated intravenously. For example, other reactionproducts of ethylene oxide and castor oil can be so used, as canreaction products of ethylene, propylene and other similar oxides withother fatty acids and the reaction products of propylene and othersimilar oxides with castor oil. Similarly, glycols other than1,2-propanediol can be used in producing the emulsions for intravenousadministration, or the glycol can be omitted, particularly if thesolubilizer is prepared to have a lower viscosity and greatercompatibility with water, by comparison with the solubilizer that isavailable under the designation CREMOPHOR EL. It is necessary only thatthe solution or emulsion be one which is physiologically acceptable andof a suitable concentration, or dilutable to a suitable concentration,for intravenous administration. An indefinitely large number of suchsolutions and emulsions will be apparent to those skilled in therelevant art from the foregoing specific disclosure. Similarly, theaqueous phase need not be 0.9 percent w/w or any other concentration ofsodium chloride. Such saline is presently favored for intravenousadministration, but other aqueous phases can also be used, so long asthe entire composition is physiologically acceptable for intravenousadministration, and, in fact, other aqueous phases may subsequently befavored.

Dosages of 4 and 10 mg per kg of body weight were used in the in vivoprocedures described above. It has not been determined that 4 mg per kgis the minimum dosage or that 10 mg per kg is the maximum. Both dosagescaused the biological consequences described above. It will beappreciated, therefore, that it is necessary only to use an effectiveamount of a purpurin or chlorin according to the invention in thedetection and treatment of tumors, preferably as small a dosage aspossible, and that the exact dosage can be determined by routineexperimentation. Both systemic administration, specifically intravenous,and local administration, i.e., as a solution in dimethyl sulfoxide orethanol, have been described above; however, it will also be appreciatedthat other methods of administration will be suitable, at least in someinstances.

Illumination of tumors containing a purpurin, a chlorin or a metalcomplex in accordance with the instant invention can be a surfaceillumination with a conventional light source, as described above, orcan be a surface illumination with a laser. The illumination can also beinto the body of a tumor, for example through optical fibers insertedthereinto.

Various changes and modification can be made from the specific detailsof the invention as described above without departing from the spiritand scope thereof as defined in the appended claims.

We claim:
 1. A composition consisting essentially of a solution in asolvent of a chlorin having the structure of any of FIGS. 24-28 or apurpurin having the structure of any of FIGS. 14-18 ##STR1## whereineach of R10 through R14 is hydrogen, and each of R1 through R9 is: H orCHO,an alkyl group other than t-butyl having from 1 to 4 carbon atoms,an alkylene group having from 2 to 4 carbon atoms, a group having theformula R₂ N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbon radicalhaving from 1 to 4 carbon atoms, wherein any carbon to carbon bond iseither a single or a double bond, and not more than one is a doublebond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atomsand the two R₃ groups can be the same or different, a group having theformula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatic hydrocarbon radicalhaving from 1 to 4 carbon atoms, wherein any carbon to carbon bond iseither a single or a double bond, and not more than one is a doublebond; A is a physiologically acceptable anion; and R₄ is an alkyl grouphaving from 1 to 2 carbon atoms and the three R₄ groups can be the sameor different, a group having the formula R₂ OH were R₂ is a bivalentaliphatic hydrocarbon radical having from 1 to 4 carbon atoms, whereinany carbon to carbon bond is either a single or a double bond, and notmore than one is a double bond, an amino acid moiety which is a part ofan amide produced by reaction between an amine function of a naturallyoccurring amino acid and a carboxyl function of the purpurin or chlorin,CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R', where R' is hydrogen or an alkylgroup other than t-butyl having from 1 to 4 carbon atoms, a monoclonalantibody moiety which is attached to the purpurin or chlorin moietythrough a carbonyl which is a part of an amide produced by reactionbetween an amine function of a monoclonal antibody and a CO₂ R' CH₂ CO₂R' or CH₂ CH₂ CO₂ R' group of the purpurin or chlorin, and wherein themoiety is of a monoclonal antibody which selectively binds to malignanttumors, or one or both of R1 and R2 can be a bivalent aliphatichydrocarbon radical having from 2 to 4 carbon atoms wherein both of thevalences of the radical are attached to the same carbon atom thereof andto a carbon atom of the chlorin or purpurin, with the proviso that notmore than one of the R1 through R9, and R14, is a group having theformula R₂ N(R₃)₂, a group having the formula R₂ N(R₄)₃ A, an amino acidmoiety or a monoclonal antibody moiety,and wherein the solutioncomprises an organic liquid, and is one which is physiologicallyacceptable and of a suitable concentration or dilutable to a suitableconcentration for intravenous administration.
 2. As a composition ofmatter, a chlorin having the structure of any of FIGS. 24, 25, 27 and28, or a purpurin having the structure of any of FIGS. 14-18, ##STR2##wherein each of R10 through R14 is hydrogen, and each of R1 through R9is: H or CHO,an alkyl group other than t-butyl having from 1 to 4 carbonatoms, an alkylene group having from 2 to 4 carbon atoms, a group havingthe formula R₂ N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbonradical having from 1 to 4 carbon atoms, wherein any carbon to carbonbond is either a single or a double bond, and not more than one is adouble bond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbonatoms and the two R₃ groups can be the same or different, a group havingthe formula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatic hydrocarbonradical having from 1 to 4 carbon atoms, wherein any carbon to carbonbond is either a single or a double bond, and not more than one is adouble bond; A is a physiologically acceptable anion; and R₄ is an alkylgroup having from 1 to 2 carbon atoms and the three R₄ groups can be thesame or different, a group having the formula R₂ OH were R₂ is abivalent aliphatic hydrocarbon radical having from 1 to 4 carbon atoms,wherein any carbon to carbon bond is either a single or a double bond,and not more than one is a double bond, an amino acid moiety which is apart of an amide produced by reaction between an amine function of anaturally occurring amino acid and a carboxyl function of the purpurinor chlorin, CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R', where R' is hydrogenor an alkyl group other than t-butyl having from 1 to 4 carbon atoms, amonoclonal antibody moiety which is attached to the purpurin or chlorinmoiety through a carbonyl which is a part of an amide produced byreaction between an amine function of a monoclonal antibody and a CO₂ R'CH₂ CO₂ R' or CH₂ CH₂ CO₂ R' group of the purpurin or chlorin, andwherein the moiety is of a monoclonal antibody which selectively bindsto malignant tumors, or one or both of R1 and R2 can be a bivalentaliphatic hydrocarbon radical having from 2 to 4 carbon atoms whereinboth of the valences of the radical are attached to the same carbon atomthereof and to a carbon atom of the chlorin or purpurin,with the provisothat not more than one of the R1 through R9, and R14, is a group havingthe formula R₂ N(R₃)₂, a group having the formula R₂ N(R₄)₃ A, an aminoacid moiety or a monoclonal antibody moiety.
 3. As a composition ofmatter, a chlorin having the structure of any of FIGS. 24-28 or apurpurin having the structure of any of FIGS. 14-18 ##STR3## whereineach of R10 through R14 is hydrogen, and each of R1 through R9 is: H orCHO,an alkyl group other than t-butyl having from 1 to 4 carbon atoms,an alkylene group having from 2 to 4 carbon atoms, a group having theformula R₂ N(R₃)₂ where R₂ is a bivalent aliphatic hydrocarbon radicalhaving from 1 to 4 carbon atoms, wherein any carbon to carbon bond iseither a single or a double bond, and not more than one is a doublebond; R₃ is hydrogen or an alkyl group having from 1 to 2 carbon atomsand the two R₃ groups can be the same or different, a group having theformula R₂ N(R₄)₃ A where R₂ is a bivalent aliphatic hydrocarbon radicalhaving from 1 to 4 carbon atoms, wherein any carbon to carbon bond iseither a single or a double bond, and not more than one is a doublebond; A is a physiologically acceptable anion; and R₄ is an alkyl grouphaving from 1 to 2 carbon atoms and the three R₄ groups can be the sameor different, a group having the formula R₂ OH were R₂ is a bivalentaliphatic hydrocarbon radical having from 1 to 4 carbon atoms, whereinany carbon to carbon bond is either a single or a double bond, and notmore than one is a double bond, an amino acid moiety which is a part ofan amide produced by reaction between an amine function of a naturallyoccurring amino acid and a carboxyl function of the purpurin or chlorin,CO₂ R', CH₂ CO₂ R' or CH₂ CH₂ CO₂ R', where R' is hydrogen or an alkylgroup other than t-butyl having from 1 to 4 carbon atoms, a monoclonalantibody moiety which is attached to the purpurin or chlorin moietythrough a carbonyl which is a part of an amide produced by reactionbetween an amine function of a monoclonal antibody and a CO₂ R' CH₂ CO₂R' or CH₂ CH₂ CO₂ R' group of the purpurin or chlorin, and wherein themoiety is of a monoclonal antibody which selectively binds to malignanttumors, or one or both of R1 and R2 can be a bivalent aliphatichydrocarbon radical having from 2 to 4 carbon atoms wherein both of thevalences of the radical are attached to the same carbon atom thereof andto a carbon atom of the chlorin or purpurin,with the proviso that notmore than one of the R1 through R9, and R14 is CHO, a group having theformula R₂ N(R₃)₂, a group having the formula R₂ N(R₄)₃ A, an amino acidmoiety or a monoclonal antibody moiety, and wherein the purpurin,chlorin or complex has a structure which is enriched in an atom whichcan be detected by nuclear magnetic resonance, said atom being presentin the purpurin, chlorin or complex in a sufficient proportion to enablethe use of nuclear magnetic resonance testing to detect small quantitiesof the purpurin, chlorin or complex.
 4. As a composition of matter, achlorin having the structure of any of FIGS. 24-28 or a purpurin havingthe structure of any of FIGS. 14-18 ##STR4## wherein each of R10 throughR14 is hydrogen, and each of R1 through R9 is: H or CHO,an alkyl groupother than t-butyl having from 1 to 4 carbon atoms, an alkylene grouphaving from 2 to 4 carbon atoms, a group having the formula R₂ N(R₃)₂where R₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4carbon atoms, wherein any carbon to carbon bond is either a single or adouble bond, and not more than one is a double bond; R₃ is hydrogen oran alkyl group having from 1 to 2 carbon atoms and the two R₃ groups canbe the same or different, a group having the formula R₂ N(R₄)₃ A whereR₂ is a bivalent aliphatic hydrocarbon radical having from 1 to 4 carbonatoms, wherein any carbon to carbon bond is either a single or a doublebond, and not more than one is a double bond; A is a physiologicallyacceptable anion; and R₄ is an alkyl group having from 1 to 2 carbonatoms and the three R₄ groups can be the same or different, a grouphaving the formula R₂ OH were R₂ is a bivalent aliphatic hydrocarbonradical having from 1 to 4 carbon atoms, wherein any carbon to carbonbond is either a single or a double bond, and not more than one is adouble bond, an amino acid moiety which is a part of an amide producedby reaction between an amine function of a naturally occurring aminoacid and a carboxyl function of the purpurin or chlorin, CO₂ R', CH₂ CO₂R' or CH₂ CH₂ CO₂ R', where R' is hydrogen or an alkyl group other thant-butyl having from 1 to 4 carbon atoms, a monoclonal antibody moietywhich is attached to the purpurin or chlorin moiety through a carbonylwhich is a part of an amide produced by reaction between an aminefunction of a monoclonal antibody and a CO₂ R' CH₂ CO₂ R' or CH₂ CH₂ CO₂R' group of the purpurin or chlorin, and wherein the moiety is of amonoclonal antibody which selectively binds to malignant tumors, or oneor both of R1 and R2 can be a bivalent aliphatic hydrocarbon radicalhaving from 2 to 4 carbon atoms wherein both of the valences of theradical are attached to the same carbon atom thereof and to a carbonatom of the chlorin or purpurin,with the proviso that not more than oneof the R1 through R9, and R14 is CHO, a group having the formula R₂N(R₃)₂, a group having the formula R₂ N(R₄)₃ A, an amino acid moiety ora monoclonal antibody moiety, and wherein the purpurin, chlorin orcomplex has a structure which is enriched in an atom which emitsionizing radiation, said atom being present in the purpurin, chlorin orcomplex in a sufficient proportion to enable the use of an instrumentwhich detects the presence of ionizing radiation to detect smallquantities of the purpurin, chlorin or complex.